Use of markers including filamin a in the diagnosis and treatment of prostate cancer

ABSTRACT

Methods for diagnosing the presence of prostate cancer in a subject are provided, such methods including the detection of levels of variety of biomarkers diagnostic of prostate cancer, including filamin A alone, or in combination with one or more additional biomarkers of prostate cancer, including, PSA, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, filamin B, and LY9. Additionally, age can be used as a predictor variable. The invention also provides methods of treating prostate cancer which rely on diagnostic information obtained based on the detection of biomarkers of prostate cancer, including filamin A alone, or in combination with one or more additional biomarkers of prostate cancer, including, PSA, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, filamin B, LY9, and/or age. Compositions in the form of kits and panels of reagents for detecting the biomarkers of the invention are also provided.

RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.14/962,966, filed on Dec. 8, 2015, which, in turn, claims priority toU.S. Provisional Application No. 62/088,931, filed on Dec. 8, 2014; U.S.Provisional Application No. 62/134,956, filed on Mar. 18, 2015; and U.S.Provisional Application No. 62/148,294, filed on Apr. 16, 2015, theentire contents of each of which are expressly incorporated herein byreference.

SEQUENCE LISTING

This specification incorporates by reference the Sequence Listingsubmitted via EFS web on Nov. 19, 2019 identified as 119992_10806, whichis 527 kb, and was created on Nov. 18, 2019. The Sequence Listing,electronically filed, does not extend beyond the scope of thespecification and does not contain new matter.

INCORPORATION BY REFERENCE

All documents cited or referenced herein and all documents cited orreferenced in the herein cited documents, together with anymanufacturer's instructions, descriptions, product specifications, andproduct sheets for any products mentioned herein or in any documentincorporated by reference herein, are hereby incorporated by reference,and may be employed in the practice of the invention.

BACKGROUND A. Field of the Invention

The invention generally relates to novel biomarkers and combinations ofbiomarkers which can be used to detect and monitor prostate cancer. Theinvention also generally relates to methods for diagnosing, monitoring,and treating prostate cancer involving the detection of biomarkers ofthe invention.

B. Background of the Invention

Prostate cancer is a leading cause of male cancer-related deaths—secondonly to lung cancer—and afflicts one out of nine men over the age of 65.According to the American Cancer Society, 241,000 new cases of prostatecancer were reported with about 30,000 prostate cancer-related deathsthat same year. Although the disease is typically diagnosed in men overthe age of 65, its impact is still significant in that the average lifespan of a man who dies from prostate cancer is reduced by nearly adecade on average. However, if prostate cancer is discovered early, 90%of the cases may be cured with surgery. Once the tumor spreads outsidethe area of the prostate gland and forms distant metastases, the diseaseis more difficult to treat. Therefore, early detection is of criticalimportance to the success of interventional therapies, and for reducingthe mortality rate associated with prostate cancer.

Prostate cancer typically develops in the various tissues of theprostate, a gland in the male reproductive system. Most prostate cancersare slow growing. However, there are also a significant number of casesper year of aggressive prostate cancers, in which the cancer cells maymetastasize from the prostate to other parts of the body, particularlyto the bones and lymph nodes. Prostate cancer may cause pain, difficultyin urinating, problems during sexual intercourse, or erectiledysfunction. Other symptoms can potentially develop during later stagesof the disease.

Currently, prostate cancer is screened using only a limited number ofdetection means, including the digital rectal exam (DRE) and/or themeasurement of the levels of prostate specific antigen (PSA). However,these approaches have an unacceptably high rate of false-positives.Indeed, most men (75%) with an elevated PSA level turn out not to haveprostate cancer as determined by subsequent confirmatory prostatebiopsies.

As such, the current screening tests are not specific enough to robustlyscreen for prostate cancer. Each year, based on the results of the DREand PSA screens, about one million prostate biopsies are performed inthe U.S. alone. Only 25% of these biopsies confirm the presence ofcancer. PSA is secreted from epithelial cells of the prostate gland andis higher in blood due to increased number of prostate epithelial cells.When prostate cancers develop, PSA levels in the blood can start toclimb. In the United States, the FDA has approved the PSA test forannual screening of prostate cancer in men of age 50 and older. PSAlevels between 4 and 10 ng/mL are considered to be suspicious andconsideration should be given to confirming the abnormal PSA with arepeat test. If indicated, a prostate biopsy is performed to obtain atissue sample for histopathological analysis. Complications—such asinfection, internal bleeding, allergic reactions, impotence, and urinaryincontinence—induced by needless biopsies and treatments injure manymore men than are potentially helped by early detection of cancers.

Indeed, the U.S. Preventative Services Task Force (USPSTF) estimatesthat about 90% of diagnosed men are treated and 2 in 1000 men willdevelop serious cardiovascular events, 1 in 1000 men will develop deepvenous thrombosis, 29 in 1000 men will develop erectile dysfunction, 18in 1000 men will develop urinary incontinence, and 1 in 1000 men willdie due to treatment. A large majority of these men would have haveremained asymptomatic for life if left untreated. As such, most cancersfound through PSA tests are not, in fact, dangerous. Nevertheless, giventhe lack of more effective predictors of prostate cancer, the fieldtakes a more conservative approach in the use of biopsies and treatment,erring on the side of precaution but risking significant harm tootherwise healthy men.

Despite the current drawbacks in prostate cancer detection, the USPSTFestimates that one life will be saved for every 1,000 men screened every1-4 years over a 10-year period. This overall outlook can be furtherimproved by limiting unnecessary biopsies with the use of improvedpre-biopsy screening methods that are associated with fewerfalse-positive results. With fewer unnecessary biopsies, fewer men willsuffer the associated biopsy complications. In addition, fewercomplications will also lead to an overall cost reduction to thehealthcare system in the management of prostate cancer.

Accordingly, there is an unmet need for improved prostate cancerscreening tools that improve the accuracy of prostate cancer detection.Molecular-based biomarkers may address this need.

SUMMARY OF THE INVENTION

In view of the fact that prostate cancer remains a life threateningdisease reaching a significant portion of the male population, thereremains a need for efficient, accurate, and rapid molecular diagnosismeans, particularly which do not suffer from a high proportion of falseresults. The development of molecular tests for the accurate detectionof prostate cancer will also lead to improved management of appropriatetherapies, and an overall improved survival rate. Thus, there remains aneed to provide an improved diagnostic test for the detection ofprostate cancer which is more reliable and accurate than PSA and othercurrent screening tests. The present invention addresses this need byproviding the use of a new biomarker, filamin A, either used alone or incombination with other markers, for the accurate and reliable detectionof prostate cancer.

The present invention is based, at least in part, on the discovery thatfilamin A is differentially regulated in prostate cancer cells. Inparticular, the invention is based on the surprising discovery thatfilamin A levels are significantly elevated in the serum of patientswith prostate cancer. Accordingly, the invention provides methods fordiagnosing and/or monitoring (e.g., monitoring of disease progression ortreatment) an oncological disease state, e.g., prostate cancer, in amammal. The invention also provides methods for treating or foradjusting treatment regimens based on diagnostic information relating tothe levels of filamin A in the serum of a subject with an oncologicaldisease state, e.g., prostate cancer. The invention further providespanels and kits for practicing the methods of the invention.

Accordingly, in one aspect, the present invention provides a method fordiagnosing the presence of prostate cancer in a subject, comprising: (a)detecting the level of filamin A in a biological sample of the subject,and (b) comparing the level of filamin A in the biological sample with apredetermined threshold value, wherein the level filamin A above thepredetermined threshold value indicates the presence of prostate cancerin the subject.

In another aspect, the invention provides a method for diagnosing thepresence of prostate cancer in a subject, comprising: (a) contacting abiological sample with a reagent that selectively binds to filamin A;(b) allowing a complex to form between the reagent and filamin A; (c)detecting the level of the complex, and (d) comparing the level of thecomplex with a predetermined threshold value, wherein the level of thecomplex above the predetermined threshold value indicates the presenceof prostate cancer in the subject.

In certain embodiments, the diagnostic method further comprisesdetecting the level of one or more additional markers of prostatecancer.

The one or more additional markers of prostate cancer can include, butis not limited to, prostate specific antigen (PSA), filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, kertin 18, keratin 19, andtubulin-beta 3. In another embodiment, the one or more additionalmarkers of prostate cancer can include age. Age can be used as acontinuous predictive biomarker. For example, increased age isassociated with higher risk of having prostate cancer. Lower age isassociated with decreased risk of having prostate cancer.

In certain other embodiments, the one or more additional markers caninclude genes that have been described in the literature as beingspecifically expressed in the prostate. These genes can include, forexample, prostate-specific membrane antigen (PSM) (Fair et al., 1997,Prostate-specific membrane antigen. Prostate 32:140-148), prostate stemcell antigen (PSCA) (Reiter et al., 1998, Prostate stem cell antigen: acell surface marker overexpressed in prostate cancer. Proc. Natl. Acad.Sci. USA 95:1735-1740), TMPRSS2 (Lin et al., 1999. Prostate-localizedand androgen-regulated expression of the membrane-bound serine proteaseTMPRSS2. Cancer Res. 59:4180-4184), PDEF (Oettgen et al., 2000, PDEF, anovel prostate epithelium-specific ETS transcription factor, interactswith the androgen receptor and activates prostate-specific antigen geneexpression. J. Biol. Chem. 275:1216-1225), prostate-specific gene-1(Herness, 2003. A novel human prostate-specific gene-1 (HPG-1):molecular cloning, sequencing, and its potential involvement in prostatecarcinogenesis. Cancer Res. 63:329-336), and even various non-codingRNA's (ncRNA's), like PCA3 (Bussemakers et al., 1999. DD3: a newprostate-specific gene, highly overexpressed in prostate cancer, CancerRes. 59:5975-5979), PCGEM1 (Srikantan et al., 2000. PCGEM1, aprostate-specific gene, is overexpressed in prostate cancer. Proc. Natl.Acad. Sci. USA 97:12216-12221) and the gene cluster P704P, P712P, andP775P (Stolk et al., 2004. P704P, P712P, and P775P: A genomic cluster ofprostate-specific genes. Prostate 60:214-226). Only a fraction of thesemarkers have been associated with prostate cancer prognosis, progressionand/or metastatic capacity and as such, their potential as valuablebiomarkers and/or therapeutic targets is largely unknown.

In one embodiment, the prostate cancer is a prostate cancercharacterized by overexpression of filamin A. In another embodiment, theprostate cancer is a prostate cancer characterized by overexpression offilamin A and overexpression of one or more additional markers selectedfrom the group consisting of filamin B, LY9, keratin 5, keratin 7,keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, orprostate specific antigen (PSA). In another embodiment, the prostatecancer is a prostate cancer characterized by overexpression of filamin Aand overexpression of one or more additional markers selected from thegroup consisting of filamin B, LY9, keratin 5, keratin 7, keratin 8,keratin 15, keratin 18, keratin 19, tubulin-beta 3, or prostate specificantigen (PSA), and increased patient age. In another embodiment, theprostate cancer is a prostate cancer characterized by overexpression offilamin A and increased patient age. In another embodiment, the prostatecancer is a prostate cancer characterized by underexpression of filaminA and overexpression of one or more additional markers selected from thegroup consisting of filamin B, LY9, keratin 5, keratin 7, keratin 8,keratin 15, keratin 18, keratin 19, tubulin-beta 3, or prostate specificantigen (PSA). In another embodiment, the prostate cancer is a prostatecancer characterized by underexpression of filamin A and overexpressionof one or more additional markers selected from the group consisting offilamin B, LY9, keratin 5, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, tubulin-beta 3, or prostate specific antigen (PSA), andincreased patient age. In another embodiment, the prostate cancer is aprostate cancer characterized by underexpression of filamin A andincreased patient age.

In certain embodiments, the biological sample can be selected from thegroup consisting of blood, serum, urine, organ tissue, biopsy tissue,feces, skin, hair, and cheek tissue.

In various embodiments, the level of filamin A can be determined by anassay, such as an immunoassay or ELISA. Other suitable assays may beemployed.

In certain embodiments, the step of determining the level of filamin Ain the biological sample can comprise (i) contacting the biologicalsample with a reagent that selectively binds to the filamin Apolypeptide to form a biomarker complex, and (ii) detecting thebiomarker complex.

In some embodiments, the reagent can be an anti-filamin A antibody thatselectively binds to at least one epitope of filamin A. In certain otherembodiments, the anti-filamin A antibody may further comprise adetectable label. In other embodiments, the method may include a furtherstep that contacts the anti-filamin A antibody/filamin A complex with asecondary antibody which selectively binds to the anti-filamin Aantibody, and which itself carries a detectable tag or label.

In certain other embodiments, the step of determining the level offilamin A in the biological sample can be based on determining theamount of filamin A mRNA in the biological sample.

In some embodiments, an amplification reaction can be used fordetermining the amount of filamin A mRNA in the biological sample. Theamplification reaction can include, but is not limited to, (a) apolymerase chain reaction (PCR); (b) a nucleic acid sequence-basedamplification assay (NASBA); (c) a transcription mediated amplification(TMA); (d) a ligase chain reaction (LCR); or (e) a strand displacementamplification (SDA).

In still other embodiments, the step of determining the level of filaminA in the biological sample can be based on a hybridization assay, whichcan include using an oligonucleotide or probe that is complementary to aportion of a filamin A mRNA to hybridize thereto, wherein theoligonucleotide further comprises a detectable label or tag.

In certain embodiments, the prostate cancer is a prostaticintraepithelial neoplasia, adenocarcinoma, small cell carcinoma, orsquamous cell carcinoma. In other embodiments, the prostate cancer canbe an androgen-dependent prostate cancer. In still other embodiments,the prostate cancer can be an androgen-independent prostate cancer. Inyet other embodiments, the prostate cancer can be an aggressive prostatecancer or a metastasized cancer. In still other embodiments, theprostate cancer can be a non-aggressive prostate cancer.

In embodiments where a diagnosis of prostate cancer is made, theinvention also contemplates administering a therapeutic anti-cancertreatment, wherein the anti-cancer treatment is selected from the groupconsisting of (a) radiation therapy, (b) chemotherapy, (c) surgery, (d)hormone therapy, (e) antibody therapy, (f) immunotherapy, (g) cytokinetherapy, (h) growth factor therapy, and (d) any combination of (a)-(h).

In various embodiments, the methods of the invention can involve firstselecting a subject suspected of having or being at risk of havingprostate cancer and obtaining a biological sample from that subjectsuspected of having or being at risk of having prostate cancer.

In still other embodiments, the diagnostic methods of the invention mayfurther comprise comparing the level of the one or more prostate cancerrelated markers in the biological sample, e.g., filamin A and one ormore of PSA, filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin15, kertin 18, keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF,HPG-1, PCA3, and PCGEM1 with the level of the one or more prostatecancer related markers in a control sample selected from the groupconsisting of: a sample obtained from the same subject at an earliertime point than the biological sample, a sample from a subject withbenign prostatic hyperplasia (BPH), a sample from a subject withnon-metastatic prostate cancer, a sample from a subject with metastaticprostate cancer, a sample from a subject with androgen sensitiveprostate cancer, a sample from a subject with androgen insensitiveprostate cancer, a sample from a subject with aggressive prostatecancer, and a sample from a subject with non-aggressive prostate cancer.

In still other embodiments, the diagnostic methods of the invention cancomprise differentiating between two prostate cancer states selectedfrom the group consisting of: normal prostate and prostate cancer,benign prostate hyperplasia and prostate cancer, benign prostatehyperplasia and normal prostate, androgen dependent and androgenindependent prostate cancer, aggressive prostate cancer andnon-aggressive prostate cancer, and metastatic prostate cancer andnon-metastatic prostate cancer.

In yet another aspect, the present invention provides a method formonitoring prostate cancer in a subject, the method comprising: (1)determining a level of filamin A in a first biological sample obtainedat a first time from a subject having prostate cancer; (2) determining alevel of filamin A in a second biological sample obtained from thesubject at a second time, wherein the second time is later than thefirst time; and (3) comparing the level of filamin A in the secondsample with the level of filamin A in the first sample, wherein a changein the level of filamin A is indicative of a change in prostate cancerstatus in the subject.

In certain embodiments, the determining steps (1) and (2) above furthercomprise determining the level of one or more additional prostate cancerrelated markers selected from the group consisting of PSA, filamin B,LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1.In certain embodiments, the determining steps (1) and (2) above furthercomprise determining the level of the patient's age.

In certain embodiments, the subject is actively treated for prostatecancer prior to obtaining the second sample. In other embodiments, thesubject is not actively treated for prostate cancer prior to obtainingthe second sample.

In certain embodiments relating to monitoring prostate cancer, theincreased level of filamin A and/or the one or more additional prostatecancer related markers in the second biological sample as compared tothe first biological sample is indicative of progression of the prostatecancer in the subject.

In certain other embodiments relating to monitoring prostate cancer, adecreased or equivalent level of filamin A and/or the one or moreadditional prostate cancer related markers in the second biologicalsample as compared to the first biological sample is indicative ofnon-progression of the prostate cancer in the subject.

In other embodiments relating to monitoring prostate cancer, wherein themethod further comprises comparing the level of the one or more prostatecancer related markers in the first biological sample or the secondbiological sample with the level of the one or more prostate cancerrelated markers in a control sample selected from the group consistingof: a normal control sample, a sample from a subject with benignprostatic hyperplasia (BPH), a sample from a subject with non-metastaticprostate cancer, a sample from a subject with metastatic prostatecancer, a sample from a subject with androgen sensitive prostate cancer,a sample from a subject with androgen insensitive prostate cancer, asample from a subject with aggressive prostate cancer, and a sample froma subject with non-aggressive prostate cancer.

In still other embodiments, any of the methods of the invention caninclude detecting the size of the prostate tumor in the subject.

In still other embodiments, any of the methods further compriseobtaining a first sample and a second sample from the subject.

In still other embodiments, the diagnostic methods of the inventionfurther comprise the step of selecting and/or administering a differenttreatment regimen for the subject based on progression of the prostatecancer in the subject.

In yet other embodiments, the diagnostic methods of the inventionfurther comprise administering a therapeutic anti-cancer based onprogression of the prostate cancer in the subject, wherein theanti-cancer treatment is selected from the group consisting of (a)radiation therapy, (b) chemotherapy, (c) surgery, (d) hormone therapy,(e) antibody therapy, (f) immunotherapy, (g) cytokine therapy, (h)growth factor therapy, and (d) any combination of (a)-(h).

In still other embodiments, the methods of the invention furthercomprise withholding an active treatment of the prostate cancer in thesubject based on non-progression of the prostate cancer in the subject.

In another aspect, the present invention provides a method of treatingprostate cancer in a subject, comprising: (a) obtaining a biologicalsample from a subject suspected of having prostate cancer, (b)submitting the biological sample to obtain diagnostic information as tothe level of filamin A, (c) administering a therapeutically effectiveamount of an anti-cancer therapy if the level of filamin A is above athreshold level.

In yet another aspect, the present invention provides a method oftreating prostate cancer in a subject, comprising: (a) obtainingdiagnostic information as to the level of filamin A in a biologicalsample, and (b) administering a therapeutically effective amount of ananti-cancer therapy if the level of filamin A is above a thresholdlevel.

In still another aspect, the present invention provides a method oftreating prostate cancer in a subject, comprising: (a) obtaining abiological sample from a subject suspected of having prostate cancer foruse in identifying diagnostic information as to the level of filamin A,(b) measuring the level of filamin A in the biological sample, (c)recommending to a healthcare provider to administer an anti-cancertherapy if the level of filamin A is above a threshold level.

In certain embodiments, the method of the invention further comprisesobtaining diagnostic information as to the level of one or moreadditional markers of prostate cancer.

In still other embodiments, the method of the invention furthercomprises obtaining diagnostic information as to the level of one ormore additional markers of prostate cancer. The one or more additionalmarkers of prostate cancer can include, but are not limited to, PSA,filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, andPCGEM1. In another embodiment, the patient's age is determined. Age canbe used as a continuous predictive variable. For example, increased ageis associated with increased risk of prostate cancer. Conversely,decreased age is associated with decreased risk of prostate cancer.

In certain other embodiments, the method of the invention involvesadministering a therapeutically effective amount of an anti-cancertherapy if the level of filamin A and at least one of the additionalmarkers of prostate cancer are above a threshold level.

In still other embodiments, the method of the invention involvesrecommending to a healthcare provider to administer an anti-cancertherapy if the level of filamin A and at least one of the additionalmarkers of prostate cancer are above a threshold level.

The biological sample of any of the methods of the invention can beobtained from the blood, serum, urine, organ tissue, biopsy tissue,feces, skin, hair, or cheek tissue, or any other suitable tissue orbodily site.

In still further embodiments, the methods of treatment of the inventioncan measure the level of filamin A as determined by immunoassay orELISA. In still other embodiments, the level of filamin A can bedetermined by (i) contacting the biological sample with a reagent thatselectively binds to the filamin A to form a biomarker complex, and (ii)detecting the biomarker complex. The reagent can be an anti-filamin Aantibody that selectively binds to at least one epitope of filamin A.

In certain other embodiments, the level of filamin A can be determinedby measuring the amount of filamin A mRNA in the biological sample. Thefilamin A mRNA level can be determine by an amplification reaction,including (a) a polymerase chain reaction (PCR); (b) a nucleic acidsequence-based amplification assay (NASBA); (c) a transcription mediatedamplification (TMA); (d) a ligase chain reaction (LCR); or (e) a stranddisplacement amplification (SDA). The level of filamin A mRNA can alsobe determined by a hybridization assay using an oligonucleotide that iscomplementary to a portion of a filamin A mRNA.

In still another aspect, the present invention relates to a kit fordetecting filamin A in a biological sample comprising at least onereagent for measuring the level of filamin A in the biological sample,and a set of instructions for measuring the level of filamin A. Thereagent can be an anti-filamin A antibody. The kit can also comprise ameans to detect the anti-filamin A antibody, such as a detectablesecondary antibody.

The kit of the invention may also include a reagent that is anoligonucleotide that is complementary to a filamin A mRNA.

The kit of the invention can also include a set of instructions whichset forth an immunoassay or ELISA for detecting the filamin A level inthe biological sample. The instruction may set forth an amplification orhybridization reaction for assaying the level of filamin A mRNA in thebiological sample. The amplification reaction can be (a) a polymerasechain reaction (PCR); (b) a nucleic acid sequence-based amplificationassay (NASBA); (c) a transcription mediated amplification (TMA); (d) aligase chain reaction (LCR); or (e) a strand displacement amplification(SDA).

In still another aspect, the present invention provides a panel for usein a method of detecting at least two markers for prostate cancer, thepanel comprising at least two detection reagents, wherein each detectionreagent is specific for the detection of at least one prostate cancermarker of a set of markers, wherein the set of markers comprises filaminA and at least one other prostate cancer related marker selected fromthe group consisting of PSA, filamin B, LY9, keratin 4, keratin 7,keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSM,PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1. In another embodiment, thepatient's age is also used as a continuous predictor variable.

In yet another aspect, the present invention provides a panel for use ina method of treating prostate cancer, the panel comprising at least twodetection reagents, wherein each detection reagent is specific for thedetection of at least one prostate cancer marker of a set of markers,wherein the set of markers comprises filamin A and at least one otherprostate cancer related marker selected from the group consisting ofPSA, filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin 15,keratin 18, keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1,PCA3, and PCGEM1. In another embodiment, the patient's age is also usedas a continuous predictor variable.

In still another aspect, the invention provides a panel for use in amethod of monitoring the treatment of prostate cancer, the panelcomprising at least two detection reagents, wherein each detectionreagent is specific for the detection of at least one prostate cancermarker of a set of markers, wherein the set of markers comprises filaminA and at least one other prostate cancer related marker selected fromthe group consisting of PSA, filamin B, LY9, keratin 4, keratin 7,keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSM,PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1. In another embodiment, thepatient's age is also used as a continuous predictor variable.

In still another aspect, the present invention relates to the use of apanel comprising a plurality of detection reagents specific fordetecting markers of prostate cancer in a method for diagnosing and/ortreating prostate cancer, wherein at least one detection reagent of thepanel is specific for detecting filamin A, and wherein the remaining oneor more detection reagents are specific for detecting a prostate cancermarker selected from the group consisting of PSA, filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1. Inanother embodiment, the patient's age is also used as a continuouspredictor variable.

In yet another aspect, the invention provides methods for diagnosing anabnormal prostate state in a subject comprising: (1) determining a levelof filamin A in combination with one or more additional prostate cancerrelated markers selected from the group consisting of PSA, filamin B,LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1 ina biological sample from the subject; and (2) comparing the level of thefilamin A and level of the one or more prostate cancer related markersin the biological sample with the corresponding levels in a normalcontrol sample, wherein an altered level of the filamin A and the one ormore prostate cancer related markers in the biological sample relativeto the normal control sample is indicative of an abnormal prostate statein the subject. In another embodiment, the patient's age is also used asa continuous predictor variable.

In certain embodiments, the one or more prostate cancer related markersis selected from the group consisting of filamin B, LY9, and keratin 19.In certain embodiments, an increased level of filamin A and at least oneor more prostate cancer related markers selected from the groupconsisting of filamin B, LY9, and keratin 19 in the biological samplerelative to a normal control sample is indicative of an abnormalprostate state in the subject. In another embodiment, the patient's ageis also used as a continuous predictor variable.

In certain embodiments, no increase in the detected level of expressionof filamin A and at least one of the prostate-cancer related markersselected from the group consisting of filamin B, LY9, and keratin 19 inthe biological sample relative to a normal control sample is indicativeof a normal prostate state in the subject. In such embodiments, levelsof one, two, or all three of filamin B, LY9, and keratin 19 can bedetected. In certain embodiments, none of the markers have increasedlevels. In another embodiment, the patient's age is also used as acontinuous predictor variable.

In certain embodiments, the method further comprises detecting the levelof prostate specific antigen (PSA) in the biological sample andpreferably further comprising comparing the level of PSA in thebiological sample to the level of PSA in a normal control sample. Incertain embodiments, an increase in the level of filamin A and at leastone or more prostate cancer related markers selected from the groupconsisting of filamin B, LY9, and keratin 19 in the biological samplerelative to the normal control sample, in combination with an increasein the level of PSA in the biological sample as compared to a normalcontrol sample has greater predictive value of the subject having anabnormal prostate state than the predictive value of a single markeralone. In certain embodiments, no increase in the detected level ofexpression of filamin A and in combination with the one or moreprostate-cancer related markers selected from the group consisting offilamin B, LY9, and keratin 19 in the biological sample relative to thenormal control sample, in further combination with a decreased or normallevel of PSA in the biological sample as compared to the level of PSA inthe normal control sample has a greater predictive value of the subjecthaving a normal prostate state than any single marker alone. In anotherembodiment, the patient's age is also used as a continuous predictorvariable.

Throughout the methods, kits, and panels of the invention, filamin A incombination with one or more of filamin B, LY9 and keratin 19 isunderstood filamin A in combination with any of filamin B; LY9; keratin19; filamin B and LY9; filamin B and keratin 19; LY9 and keratin 19; orfilamin B, LY9, and keratin 19. In one embodiment, the methods, kits andpanels of the invention include filamin A in combination with filamin B,and Keratin 19 (KRT19). In one embodiment, the methods, kits and panelsof the invention include filamin A in combination with filamin B,Keratin 19 (KRT19), and a determination of the patient's age.

Further, the invention contemplates that filamin A may be combined withany one, two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen,nineteen, twenty or more other prostate cancer related markers in anycombinations, including any of PSA, filamin B, LY9, keratin 4, keratin7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSM,PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1. In one embodiment, themethods, kits and panels of the invention include filamin A incombination with filamin B, and Keratin 19 (KRT19).

In certain embodiments of the invention, the abnormal prostate state isprostate cancer.

In certain embodiments of the invention, the prostate cancer isandrogen-dependent prostate cancer. In certain embodiments of theinvention, the prostate cancer is androgen-independent prostate cancer.In certain embodiments of the invention, the prostate cancer isaggressive prostate cancer. In certain embodiments of the invention, theprostate cancer is non-aggressive prostate cancer.

In certain embodiments of the invention, the abnormal prostate state isbenign prostate hyperplasia.

In another aspect, the invention provides a method for identifying asubject as being at increased risk for developing prostate cancer, themethod comprising: (1) determining a level of filamin A in combinationwith one or more additional prostate cancer related markers selectedfrom the group consisting of PSA, filamin B, LY9, keratin 4, keratin 7,keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSM,PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1 in a biological sample fromthe subject; and (2) comparing the level of filamin A and the one ormore prostate cancer related markers in the biological sample with thelevel of the markers in a normal control sample, wherein an alteredlevel of the markers in the biological sample relative to the controlsample is indicative of an increased risk for developing prostate cancerin the subject. In another embodiment, the patient's age is also used asa continuous predictor variable.

In certain embodiments, filamin A detection is combined with detectionof one or more prostate cancer related markers selected from the groupconsisting of filamin B, LY9, and keratin 19. In certain embodiments, anincreased level of filamin A and one or more prostate cancer relatedmarkers selected from the group consisting of filamin B, LY9, andkeratin 19 in the biological sample relative to the normal controlsample is indicative of an increased risk for developing prostate cancerin the subject. In certain embodiments, no increase in the detectedlevel of expression of filamin A and of each of the one or moreprostate-cancer related markers selected from the group consisting offilamin B, LY9, and keratin 19 in the biological sample relative to thenormal control sample is indicative of no increased risk for developingprostate cancer in the subject. In another embodiment, the patient's ageis also used as a continuous predictor variable.

In certain embodiments, the method further comprises detecting the levelfilamin A together with prostate specific antigen (PSA) in thebiological sample. In this embodiment, the method involves comparing thelevels of filamin A and PSA in the biological sample to thecorresponding levels in a normal control sample. In certain embodiments,the method further comprises measuring the levels of one or moreadditional prostate cancer related markers selected from the groupconsisting of filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin15, keratin 18, keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF,HPG-1, PCA3, and PCGEM1 in the biological sample relative to the normalcontrol sample, thereby increasing the predictive value of developingprostate cancer in the subject. In certain other embodiments, noincrease in the detected level of expression of filamin A and PSA in thebiological sample relative to the normal sample is indicative of havingno increased risk for developing prostate cancer. In still furtherembodiments, no increase in the detected levels of one or moreadditional prostate cancer related markers selected from the groupconsisting of filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin15, keratin 18, keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF,HPG-1, PCA3, and PCGEM1 is indicative of no increased risk of prostatecancer, which has a greater predictive value of no increased risk ofprostate cancer than evaluating filamin A and/or PSA alone. In anotherembodiment, the patient's age is also used as a continuous predictorvariable.

In certain embodiments of the diagnostic or prognostic methods of theinvention, the method of diagnosis of the invention is carried out onthe basis of filamin A, optionally on the additional basis of PSA, andstill optionally on the basis of one or more additional prostate cancerrelated markers selected from the group consisting of filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1. Incertain embodiments, the one or more additional prostate cancer relatedmarkers is selected from the group consisting of keratin 7, keratin 8,and keratin 15. In certain other embodiments, the one or more additionalprostate cancer related markers is selected from the group consisting ofkeratin 7 and keratin 15. In certain other embodiments, the one or moreadditional prostate cancer markers is selected from the group consistingof keratin 7, 15, and 19. In another embodiment, the patient's age isalso used as a continuous predictor variable.

In certain embodiments, the control sample for filamin A and PSA is thesame control sample as for the one or more additional prostate cancerrelated markers of the invention. In certain embodiments, the controlsample for the filamin A and PSA is different from the control sampleused for the one or more additional prostate cancer related markers ofthe invention. In still other embodiments, the control sample for thefilamin A is different from the control sample used for PSA, which areeach also different from the control sample used to measure the one ormore additional prostate cancer markers.

In certain embodiments of the diagnostic methods of the invention,wherein one or more prostate cancer related markers that are combinedwith filamin A is selected from the group consisting of filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1, anincreased level of one or more of the prostate cancer related markers inthe biological sample relative to a normal control sample is indicativeof an abnormal prostate state in the subject. In certain embodiments ofthe diagnostic methods of the invention, wherein one or more additionalprostate cancer related markers is selected from the group consisting offilamin B, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, andPCGEM1, a decreased or normal level of one or more of the additionalprostate cancer related markers in the biological sample relative to anormal control sample is indicative of an abnormal prostate state in thesubject. In certain embodiments of the diagnostic methods of theinvention, wherein one or more prostate cancer related markers isselected from the group consisting of filamin B, LY9, keratin 4, keratin7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSM,PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1, an increased level of oneor more of the additional prostate cancer related markers in thebiological sample relative to a normal control sample is indicative of anormal prostate state in the subject. In certain embodiments of thediagnostic methods of the invention, wherein one or more prostate cancerrelated markers is selected from the group consisting of filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1, adecreased or normal level of one or more of the prostate cancer relatedmarkers in the biological sample relative to a normal control sample isindicative of a normal prostate state in the subject. In anotherembodiment, the patient's age is also used as a continuous predictorvariable.

In certain embodiments of the prognostic methods of the invention,wherein the one or more additional prostate cancer related markers thatare combined with filamin A is selected from the group consisting ofPSA, filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin 15,keratin 18, keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1,PCA3, and PCGEM1, an increased level of one or more of the additionalprostate cancer related markers in the biological sample relative to anormal control sample—in addition to increased filamin A—is indicativeof an increased risk of developing prostate cancer in the subject. Incertain embodiments of the prognostic methods of the invention, whereinone or more additional prostate cancer related markers is selected fromthe group consisting of PSA, filamin B, LY9, keratin 4, keratin 7,keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSM,PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1, a decreased or normallevel of one or more of the additional prostate cancer related markersin the biological sample relative to a normal control sample—in additionto decreased filamin A—is indicative of a decreased risk of developingprostate cancer in the subject. In certain embodiments of the prognosticmethods of the invention, wherein one or more additional prostate cancerrelated markers is selected from the group consisting of PSA, filamin B,LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1,an increased level of one or more of the prostate cancer related markersin the biological sample relative to a normal control sample—in additionto increased filamin A—is indicative of increased risk of developingprostate cancer in the subject. In certain embodiments of the prognosticmethods of the invention, wherein one or more additional prostate cancerrelated markers is selected from the group consisting of PSA, filamin B,LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1, adecreased or normal level of one or more of the additional prostatecancer related markers in the biological sample—in addition to decreasedfilamin A—relative to a normal control sample is indicative of noincreased risk of developing prostate cancer in the subject. In anotherembodiment, the patient's age is also used as a continuous predictorvariable.

In certain embodiments that involve the detection of both filamin A andPSA, the method of the invention can comprise detection of one or moreadditional prostate cancer related markers that are selected from thegroup consisting of filamin B, LY9, keratin 4, keratin 7, keratin 8,keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2,PDEF, HPG-1, PCA3, and PCGEM1. In certain embodiments, an increase inthe level of both filamin A and PSA in combination with an increase inthe level of at least one of the additional prostate cancer relatedmarkers selected from the group consisting of filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1 is indicative of anabnormal prostate state in the subject wherein the method has greaterdiagnostic or predictive value than the value of any of the individualmarkers alone. In certain other embodiments, a decrease in the level ofboth filamin A and PSA in combination with a decrease in the level of atleast one of the additional prostate cancer related markers selectedfrom the group consisting of filamin B, LY9, keratin 4, keratin 7,keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSM,PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1 is indicative of a normalprostate state in the subject wherein the method has greater diagnosticor predictive value than the value of any of the individual markersalone. In another embodiment, the patient's age is also used as acontinuous predictor variable.

In various embodiments of any of the diagnostic or prognostic methods ofthe invention, the method may further comprise comparing the level ofthe one or more prostate cancer related markers in the biological samplewith the level of the one or more prostate cancer related markers in acontrol sample selected from the group consisting of: a sample obtainedfrom the same subject at an earlier time point than the biologicalsample, a sample from a subject with benign prostatic hyperplasia (BPH),a sample from a subject with non-metastatic prostate cancer, a samplefrom a subject with metastatic prostate cancer, a sample from a subjectwith androgen sensitive prostate cancer, a sample from a subject withandrogen insensitive prostate cancer, a sample from a subject withaggressive prostate cancer, and a sample from a subject withnon-aggressive prostate cancer. In such embodiments, comparison with oneor more additional control sample can facilitate differentiating betweentwo prostate cancer states selected from the group consisting of: normalprostate and prostate cancer, benign prostate hyperplasia and prostatecancer, benign prostate hyperplasia and normal prostate, androgendependent and androgen independent prostate cancer, aggressive prostatecancer and non-aggressive prostate cancer, and metastatic prostatecancer and non-metastatic prostate cancer; or differentiating betweenany two or more of normal prostate, prostate cancer, benign prostatehyperplasia, androgen dependent prostate cancer, androgen independentprostate cancer, aggressive prostate cancer, non-aggressive prostatecancer, metastatic prostate cancer, and non-metastatic prostate cancer.

In certain embodiments of the invention, when a tumor is present, themethod further comprises detecting the size of the prostate tumor in thesubject.

In certain embodiments of the diagnostic and prognostic methods theinvention, the method further comprises obtaining a sample from asubject.

In certain embodiments of the diagnostic and prognostic methods theinvention, the method further comprises selecting a subject who has oris suspected of having prostate cancer.

In certain embodiments of the invention, the method further comprisesselecting a treatment regimen for the subject based on the level of theone or more prostate cancer markers. In certain embodiments of theinvention, the method further comprises treating the subject with a atreatment regimen based on the level of the one or more prostate cancermarkers. In certain embodiments, a treatment regimen comprises one ormore treatments selected from the group consisting of surgery,radiation, hormone therapy, antibody therapy, growth factor therapy,cytokine therapy, and chemotherapy.

Where applicable or not specifically disclaimed, any one of theembodiments described herein are contemplated to be able to combine withany other one or more embodiments, even though the embodiments aredescribed under different aspects of the invention.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIG. 1: Schematic representing the underlying principles of theInterrogative Platform Technology (a.k.a. Interrogative Biology™)provided in WO2012119129, the entire contents of which are incorporatedherein by reference.

FIG. 2A, FIG. 2B, and FIG. 2C: Causal associations of keratins,including (FIG. 2A) KRT4, KRT8, KRT15 and (FIG. 2B) KRT18 and (FIG. 2C)KRT19 in human prostate cancer cells as inferred by the InterrogativePlatform Technology.

FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D: Mechanistic insight intoregulation of keratins by mitochondrial function inferred by theInterrogative Platform Technology. (FIG. 3A) KRT8-KRT15 association isabolished upon ubidecaronone treatment. Note change of direction ofarrow between and positions of KRT7 and KRT15 before treatment (FIG. 3A)and after treatment (FIG. 3B). (FIG. 3C) Tubulin-beta 3 interacts with anumber of proteins. (FIG. 3D) Expression levels of keratin 19 inbiological samples from subjects with prostate cancer or controlsamples.

FIG. 4: Inference of filamin B (FLNB) as a hub of activity in prostatecancer and as a biomarker using the Interrogative Platform Technologyprovided in WO2012119129.

FIG. 5: Portion of an inference map showing filamin B is connecteddirectly to LY9, which is, in turn, connected to at least one othermarker.

FIG. 6A and FIG. 6B: Validation of filamin B levels in human serumsamples. Levels of filamin B (FIG. 6A) and PSA (FIG. 6B) were elevatedin prostate cancer samples when compared to normal serum. Datarepresents percent average change, with normal donors set to 100% on alog scale.

FIG. 7: Validation of LY9 levels in human serum samples. Levels of LY9were elevated in prostate cancer samples when compared to normal serum.Data represents percent average change, with normal donors set to 100%on a log scale.

FIG. 8A, FIG. 8B, and FIG. 8C: Validation of filamin B (FIG. 8A), LY9(FIG. 8B), and PSA (FIG. 8C) levels in human serum samples. Data areshown as ng/ml of the marker in serum.

FIG. 9A and FIG. 9B: (FIG. 9A) ROC curve analysis of sensitivity andfalse positive rate (FPR) of PSA, FLNB and the combination of PSA andFLNB and (FIG. 9B) area under the curve values (AUC) calculated based onthe analysis. The combination of PSA and FLNB was more sensitive thaneither marker alone.

FIG. 10A and FIG. 10B: (FIG. 10A) ROC curve analysis of PSA, FLNB, LY9and combinations of PSA, FLNB, and LY9 using linear and (FIG. 10B)non-linear scoring functions. The combination of PSA, LY9, and FLNB wasmore sensitive than any marker alone.

FIG. 11: Medical annotations for the serum samples used in connectionwith filamin A ELISA, as described in the Example 13.

FIG. 12: Medical annotations for the serum samples used in connectionwith keratin 19 ELISA, as described in the Example 13.

FIG. 13: Filamin A protein levels in serum from patients with andwithout prostate cancer as determined by ELISA.

FIG. 14: Keratin 19 protein levels in serum from patients with andwithout prostate cancer as determined by ELISA.

FIG. 15: ROC curve analysis for filamin A (FLNA) and filamin C (FLNC)and the combination (FLNA/C), as per Example 14.

FIG. 16: ROC curve analysis for keratin 18 (KRT18) and kertain 19(KRT19) and the combination (KRT18/19), as per Example 14.

FIG. 17: AUC summary of AUC for PSA, Age, Filamin A (FLNA), Filamin B(FLNB), Keratin 19 (KRT19), and combinations thereof.

FIG. 18: PCA versus Else: Sensitivity match PSA.

FIG. 19: Predicted probability distribution for PCA versus Else.

FIG. 20: Accuracy analysis for PCA versus Else.

FIG. 21: Super High Gleason versus Else.

FIG. 22: Predicted probability distrubtion for Super High Gleason (8-10)versus Else.

FIG. 23: Acuracy Analysis for Super High Gleason (8-10) versus Else.

FIG. 24: High Gleason Versus Else.

FIG. 25: Predicted probability distrubtion for High Gleason (7 andabove) versus Else.

FIG. 26: Accuracy Analysis for High Gleason (7 and above) versus Else.

FIG. 27: Prostate Cancer (PCA) versus Benign Prostatic Hyperplasia (BPH)Sensitivity.

FIG. 28: Predicted probability distribution for Prostate Cancer (PCA)versus Benign Prostatic Hyperplasia (BPH).

FIG. 29: Accuracy analysis for Prostate Cancer (PCA) versus BenignProstatic Hyperplasia (BPH).

FIG. 30: FLNA, FLNB, and KRT19 expression in prostate cancer cells invitro.

FIG. 31: FLNA, FLNB, and KRT19 expression in prostate cancer cells invitro.

FIG. 32: Secretion of FLNA, FLNB, and KRT19 from prostate cancer cellsin vitro.

FIG. 33: Transcriptional regulation of FLNA, FLNB, KRT19, and PSAexpression by prostate-relevant stimuli of hypoxia (1% oxygen) in vitro.

FIG. 34: Transcriptional regulation of FLNA, FLNB, KRT19, and PSAexpression by prostate-relevant stimuli of TNFα (10 ng/mL) in vitro.

FIG. 35: Transcriptional regulation of FLNA, FLNB, KRT19, and PSAexpression by prostate-relevant stimuli of R1881 (1 nM) in vitro.

FIG. 36: Assessment of plasma FLNA and FLNB levels as biomarkers forprostate cancer.

DETAILED DESCRIPTION OF THE INVENTION A. Overview

The identification of tumor markers or antigens associated with prostatecancer has stimulated considerable interest as promising tools for thescreening, diagnosis, prognosis, clinical management and potentialtreatment of prostate cancer, and in particular, early detection ofprostate cancer. Indeed, early detection mitigates the risk that thecancer will metastasize. Non-metastasized, local prostate tumors canoften be cured by radical prostatectomy or radiation therapy, howeverfor patients with distantly spread disease, no curative treatment isavailable. This emphasizes the need for new prostate (cancer) specificdiagnostic tools that may improve the chances for accurate earlydetection.

While some prostate-specific markers are known, e.g., prostate-specificantigen and prostate stem cell antigen, very few biomarkers are inwidespread or routine use as molecular diagnostics for prostate cancer.Accordingly, there remains a need for efficient, accurate, and rapidmolecular diagnosis means, particularly which do not suffer from a highproportion of false results. The development of molecular tests for theaccurate detection of prostate cancer will also lead to improvedmanagement of appropriate therapies, and an overall improved survivalrate. Thus, there remains a need to provide an improved diagnostic testfor the detection of prostate cancer which is more reliable and accuratethan PSA and other current screening tests. The present inventionaddresses this need by providing the use of a new biomarker, filamin A,either used alone or in combination with other markers, for the accurateand reliable detection of prostate cancer.

As presently described herein, the invention at hand is based, at leastin part, on the discovery that filamin A (“FLNA”) is differentiallyregulated in prostate cancer cells and serves as a useful biomarker ofprostate cancer. In one embodiment, filamin A can serve as a usefuldiagnostic biomarker to predict and/or detect the presence of prostatecancer in a subject. In another embodiment, filamin A can serve as auseful prognostic biomarker, serving to inform on the likely progressionof prostate cancer in a subject with or without treatment. In stillanother embodiment, filamin A can serve as a useful predictive biomarkerfor helping to assess the likely response of prostate cancer to aparticular treatment. Accordingly, the invention provides methods thatuse biomarkers, e.g., filamin A, in the diagnosis of prostate cancer(e.g., prediction of the presence of prostate cancer in a subject), inthe prognosis of prostate cancer (e.g., prediction of the course oroutcome of prostate cancer with or without treatment), and in theassessment of therapies intended to treat prostate cancer (i.e., filaminA as a theragnostic or predictive marker). The invention furtherprovides compositions of matter (e.g., oligonucleotide probes specificfor filamin A mRNA, antibodies specific for filamin A, therapeuticagents that target filamin A), including panels comprising binding ordetection reagents specific for filamin A and optionally otherbiomarkers for use in the methods of the invention, as well as kits forpracticing the methods of the invention.

The following is a detailed description of the invention provided to aidthose skilled in the art in practicing the present invention. Those ofordinary skill in the art may make modifications and variations in theembodiments described herein without departing from the spirit or scopeof the present invention. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. The terminology used in the description of the invention hereinis for describing particular embodiments only and is not intended to belimiting of the invention. All publications, patent applications,patents, figures and other references mentioned herein are expresslyincorporated by reference in their entirety.

Although any methods and materials similar or equivalent to thosedescribed herein can also be used in the practice or testing of thepresent invention, the preferred methods and materials are nowdescribed. All publications mentioned herein are incorporated herein byreference to disclose and described the methods and/or materials inconnection with which the publications are cited.

B. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references, the entiredisclosures of which are incorporated herein by reference, provide oneof skill with a general definition of many of the terms (unless definedotherwise herein) used in this invention: Singleton et al., Dictionaryof Microbiology and Molecular Biology (2^(nd) ed. 1994); The CambridgeDictionary of Science and Technology (Walker ed., 1988); The Glossary ofGenetics, 5^(th) Ed., R. Rieger et al. (eds.), Springer Verlag (1991);and Hale & Marham, the Harper Collins Dictionary of Biology (1991).Generally, the procedures of molecular biology methods described orinherent herein and the like are common methods used in the art. Suchstandard techniques can be found in reference manuals such as forexample Sambrook et al., (2000, Molecular Cloning—A Laboratory Manual,Third Edition, Cold Spring Harbor Laboratories); and Ausubel et al.,(1994, Current Protocols in Molecular Biology, John Wiley & Sons,New-York).

The following terms may have meanings ascribed to them below, unlessspecified otherwise. However, it should be understood that othermeanings that are known or understood by those having ordinary skill inthe art are also possible, and within the scope of the presentinvention. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In the case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

As used herein, the singular forms “a”, “and”, and “the” include pluralreferences unless the context clearly dictates otherwise. All technicaland scientific terms used herein have the same meaning.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein can be modified by theterm about.

As used herein, the term “age” refers to the length of time that asubject has been alive. For example, the age of a subject is calculatedfrom the date of birth of the subject to the current date. Age can beused as a continuous predictive variable for the presence of prostatecancer. For example, increased age is associated with increased risk ofprostate cancer. Conversely, decreased age is associated with decreasedrisk of prostate cancer. Similarly, age can be used as a continuouspredictive variable for the stage, or category, of the prostate cancer.For example, age can be used as a continuous predictive variable for theGleason score of the prostate cancer.

As used herein, the term “amplification” refers to any known in vitroprocedure for obtaining multiple copies (“amplicons”) of a targetnucleic acid sequence or its complement or fragments thereof. In vitroamplification refers to production of an amplified nucleic acid that maycontain less than the complete target region sequence or its complement.Known in vitro amplification methods include, e.g.,transcription-mediated amplification, replicase-mediated amplification,polymerase chain reaction (PCR) amplification, ligase chain reaction(LCR) amplification and strand-displacement amplification (SDA includingmultiple strand-displacement amplification method (MSDA)).Replicase-mediated amplification uses self-replicating RNA molecules,and a replicase such as Q-β-replicase (e.g., Kramer et al., U.S. Pat.No. 4,786,600). PCR amplification is well known and uses DNA polymerase,primers and thermal cycling to synthesize multiple copies of the twocomplementary strands of DNA or cDNA (e.g., Mullis et al., U.S. Pat.Nos. 4,683,195, 4,683,202, and 4,800,159). LCR amplification uses atleast four separate oligonucleotides to amplify a target and itscomplementary strand by using multiple cycles of hybridization,ligation, and denaturation (e.g., EP Pat. App. Pub. No. 0 320 308). SDAis a method in which a primer contains a recognition site for arestriction endonuclease that permits the endonuclease to nick onestrand of a hemimodified DNA duplex that includes the target sequence,followed by amplification in a series of primer extension and stranddisplacement steps (e.g., Walker et al., U.S. Pat. No. 5,422,252). Twoother known strand-displacement amplification methods do not requireendonuclease nicking (Dattagupta et al., U.S. Pat. Nos. 6,087,133 and6,124,120 (MSDA)). Those skilled in the art will understand that theoligonucleotide primer sequences of the present invention may be readilyused in any in vitro amplification method based on primer extension by apolymerase. (see generally Kwoh et al., 1990, Am. Biotechnol. Lab.8:14-25 and (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86,1173-1177; Lizardi et al., 1988, BioTechnology 6:1197-1202; Malek etal., 1994, Methods Mol. Biol., 28:253-260; and Sambrook et al., 2000,Molecular Cloning—A Laboratory Manual, Third Edition, CSH Laboratories).As commonly known in the art, the oligos are designed to bind to acomplementary sequence under selected conditions.

As used herein, the term “antigen” refers to a molecule, e.g., apeptide, polypeptide, protein, fragment, or other biological moiety,which elicits an antibody response in a subject, or is recognized andbound by an antibody.

As used herein, the term “area under the curve” or “AUC” refers to thearea under the curve in a plot of sensitivity versus specificity. Forexample, see FIGS. 18, 21, 24, and 27. In one embodiment, the AUC for abiomarker, or combination of biomarkers, of the invention is 0.5. Inanother embodiment, the AUC for a biomarker, or combination ofbiomarkers, of the invention is 0.6. In another embodiment, the AUC fora biomarker, or combination of biomarkers, of the invention is 0.7. Inanother embodiment, the AUC for a biomarker, or combination ofbiomarkers, of the invention is 0.8. In another embodiment, the AUC fora biomarker, or combination of biomarkers, of the invention is 0.9. Inanother embodiment, the AUC for a biomarker, or combination ofbiomarkers, of the invention is 1.0. In specific embodiments, the AUCfor a biomarker, or combination of biomarkers, of the invention is 0.5,0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62,0.63, 0.64, 3.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74,0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86,0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98,0.99 or 1.0. In one embodiment, the AUC for a biomarker, or combinationof biomarkers, of the invention is at least 0.5. In another embodiment,the AUC for a biomarker, or combination of biomarkers, of the inventionis at least 0.6. In another embodiment, the AUC for a biomarker, orcombination of biomarkers, of the invention is at least 0.7. In anotherembodiment, the AUC for a biomarker, or combination of biomarkers, ofthe invention is at least 0.8. In another embodiment, the AUC for abiomarker, or combination of biomarkers, of the invention is at least0.9. In another embodiment, the AUC for a biomarker, or combination ofbiomarkers, of the invention is at least 1.0. In specific embodiments,the AUC for a biomarker, or combination of biomarkers, of the inventionis at least 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59,0.6, 0.61, 0.62, 0.63, 0.64, 3.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71,0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83,0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95,0.96, 0.97, 0.98, 0.99 or 1.0

As used herein, the term “biomarker” is understood to mean a measurablecharacteristic that reflects in a quantitative or qualitative manner thephysiological state of an organism. The physiological state of anorganism is inclusive of any disease or non-disease state, e.g., asubject having prostate cancer or a subject who is otherwise healthy.Said another way, biomarkers are characteristics that can be objectivelymeasured and evaluated as indicators of normal processes, pathogenicprocesses, or pharmacologic responses to a therapeutic intervention.Biomarkers can be clinical parameters (e.g., age, performance status),laboratory measures (e.g., molecular biomarkers, such as prostatespecific antigen), imaging-based measures, or genetic or other moleculardeterminants, such as phosphorylation or acetylation state of a proteinmarker, methylation state of nucleic acid, or any other detectablemolecular modification to a biological molecule. Examples of biomarkersinclude, for example, polypeptides, peptides, polypeptide fragments,proteins, antibodies, hormones, polynucleotides, RNA or RNA fragments,microRNA (miRNAs), lipids, polysaccharides, and other bodilymetabolites. Other examples of biomarkers include the age of thepatient.

Preferably, a biomarker of the present invention is modulated (e.g.,increased or decreased level) in a biological sample from a subject or agroup of subjects having a first phenotype (e.g., having a disease) ascompared to a biological sample from a subject or group of subjectshaving a second phenotype (e.g., not having the disease, e.g., acontrol). A biomarker may be differentially present at any level, but isgenerally present at a level that is increased relative to normal orcontrol levels by at least 5%, by at least 10%, by at least 15%, by atleast 20%, by at least 25%, by at least 30%, by at least 35%, by atleast 40%, by at least 45%, by at least 50%, by at least 55%, by atleast 60%, by at least 65%, by at least 70%, by at least 75%, by atleast 80%, by at least 85%, by at least 90%, by at least 95%, by atleast 100%, by at least 110%, by at least 120%, by at least 130%, by atleast 140%, by at least 150%, or more; or is generally present at alevel that is decreased relative to normal or control levels by at least5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%,by at least 30%, by at least 35%, by at least 40%, by at least 45%, byat least 50%, by at least 55%, by at least 60%, by at least 65%, by atleast 70%, by at least 75%, by at least 80%, by at least 85%, by atleast 90%, by at least 95%, or by 100% (i.e., absent). A biomarker ispreferably differentially present at a level that is statisticallysignificant (e.g., a p-value less than 0.05 and/or a q-value of lessthan 0.10 as determined using either Welch's T-test or Wilcoxon'srank-sum Test).

As used herein, the term “biopsy” or “biopsy tissue” refers to a sampleof tissue (e.g., prostate tissue) that is removed from a subject for thepurpose of determining if the sample contains cancerous tissue. Thebiopsy tissue is then examined (e.g., by microscopy) for the presence orabsence of cancer.

As used herein, the term “complementary” refers to the broad concept ofsequence complementarity between regions of two nucleic acid strands orbetween two regions of the same nucleic acid strand. It is known that anadenine residue of a first nucleic acid region is capable of formingspecific hydrogen bonds (“base pairing”) with a residue of a secondnucleic acid region which is antiparallel to the first region if theresidue is thymine or uracil. Similarly, it is known that a cytosineresidue of a first nucleic acid strand is capable of base pairing with aresidue of a second nucleic acid strand which is antiparallel to thefirst strand if the residue is guanine. A first region of a nucleic acidis complementary to a second region of the same or a different nucleicacid if, when the two regions are arranged in an antiparallel fashion,at least one nucleotide residue of the first region is capable of basepairing with a residue of the second region. Preferably, the firstregion comprises a first portion and the second region comprises asecond portion, whereby, when the first and second portions are arrangedin an antiparallel fashion, at least about 50%, and preferably at leastabout 75%, at least about 90%, or at least about 95% of the nucleotideresidues of the first portion are capable of base pairing withnucleotide residues in the second portion. More preferably, allnucleotide residues of the first portion are capable of base pairingwith nucleotide residues in the second portion.

The term “control sample,” as used herein, refers to any clinicallyrelevant comparative sample, including, for example, a sample from ahealthy subject not afflicted with an oncological disorder, e.g.,prostate cancer, or a sample from a subject from an earlier time point,e.g., prior to treatment, an earlier tumor assessment time point, at anearlier stage of treatment. A control sample can be a purified sample,protein, and/or nucleic acid provided with a kit. Such control samplescan be diluted, for example, in a dilution series to allow forquantitative measurement of levels of analytes, e.g., markers, in testsamples. A control sample may include a sample derived from one or moresubjects. A control sample may also be a sample made at an earlier timepoint from the subject to be assessed. For example, the control samplecould be a sample taken from the subject to be assessed before the onsetof an oncological disorder, e.g., prostate cancer, at an earlier stageof disease, or before the administration of treatment or of a portion oftreatment. The control sample may also be a sample from an animal model,or from a tissue or cell line derived from the animal model ofoncological disorder, e.g., prostate cancer. The level of activity orexpression of one or more markers (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 ormore markers) in a control sample consists of a group of measurementsthat may be determined, e.g., based on any appropriate statisticalmeasurement, such as, for example, measures of central tendencyincluding average, median, or modal values. Different from a control ispreferably statistically significantly different from a control.

As used herein, “changed as compared to a control” sample or subject isunderstood as having a level of the analyte or diagnostic or therapeuticindicator (e.g., marker) to be detected at a level that is statisticallydifferent than a sample from a normal, untreated, or abnormal statecontrol sample. Changed as compared to control can also include adifference in the rate of change of the level of one or more markersobtained in a series of at least two subject samples obtained over time.Determination of statistical significance is within the ability of thoseskilled in the art and can include any acceptable means for determiningand/or measuring statistical significance, such as, for example, thenumber of standard deviations from the mean that constitute a positiveor negative result, an increase in the detected level of a biomarker ina sample (e.g., prostate cancer sample) versus a control or healthysample, wherein the increase is above some threshold value, or adecrease in the detected level of a biomarker in a sample (e.g.,prostate cancer sample) versus a control or healthy sample, wherein thedecrease is below some threshold value. The threshold value can bedetermine by any suitable means by measuring the biomarker levels in aplurality of tissues or samples known to have a disease, e.g., prostatecancer, and comparing those levels to a normal sample and calculating astatistically significant threshold value.

The term “control level” refers to an accepted or pre-determined levelof a marker in a subject sample. A control level can be a range ofvalues. Marker levels can be compared to a single control value, to arange of control values, to the upper level of normal, or to the lowerlevel of normal as appropriate for the assay.

In one embodiment, the control is a standardized control, such as, forexample, a control which is predetermined using an average of the levelsof expression of one or more markers from a population of subjectshaving no cancer, especially subjects having no prostate cancer. Instill other embodiments of the invention, a control level of a marker isthe level of the marker in a non-cancerous sample(s) derived from thesubject having cancer. For example, when a biopsy or other medicalprocedure reveals the presence of cancer in one portion of the tissue,the control level of a marker may be determined using the non-affectedportion of the tissue, and this control level may be compared with thelevel of the marker in an affected portion of the tissue.

In certain embodiments, the control can be from a subject, or apopulation of subject, having an abnormal prostate state. For example,the control can be from a subject suffering from benign prostatehyperplasia (BPH), androgen sensitive prostate cancer, androgeninsensitive or resistant prostate cancer, aggressive prostate cancer,non-aggressive prostate cancer, metastatic prostate cancer, ornon-metastatic prostate cancer. It is understood that not all markerswill have different levels for each of the abnormal prostate stateslisted. It is understood that a combination of marker levels may be mostuseful to distinguish between abnormal prostate states, possibly incombination with other diagnostic methods. Further, marker levels inbiological samples can be compared to more than one control sample(e.g., normal, abnormal, from the same subject, from a populationcontrol). Marker levels can be used in combination with other signs orsymptoms of an abnormal prostate state to provide a diagnosis for thesubject.

A control can also be a sample from a subject at an earlier time point,e.g., a baseline level prior to suspected presence of disease, beforethe diagnosis of a disease, at an earlier assessment time point duringwatchful waiting, before the treatment with a specific agent (e.g.,chemotherapy, hormone therapy) or intervention (e.g., radiation,surgery). In certain embodiments, a change in the level of the marker ina subject can be more significant than the absolute level of a marker,e.g., as compared to control.

As used herein, “detecting”, “detection”, “determining”, and the likeare understood to refer to an assay performed for identification offilamin A and/or an additional one or more specific markers in a sample,e.g., one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 or more) markersselected from the group consisting of PSA, filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1. The amount ofmarker expression or activity detected in the sample can be none orbelow the level of detection of the assay or method.

As used herein, the term “DNA” or “RNA” molecule or sequence (as well assometimes the term “oligonucleotide”) refers to a molecule comprisedgenerally of the deoxyribonucleotides adenine (A), guanine (G), thymine(T) and/or cytosine (C). In “RNA”, T is replaced by uracil (U).

The terms “disorders”, “diseases”, and “abnormal state” are usedinclusively and refer to any deviation from the normal structure orfunction of any part, organ, or system of the body (or any combinationthereof). A specific disease is manifested by characteristic symptomsand signs, including biological, chemical, and physical changes, and isoften associated with a variety of other factors including, but notlimited to, demographic, environmental, employment, genetic, andmedically historical factors. Certain characteristic signs, symptoms,and related factors can be quantitated through a variety of methods toyield important diagnostic information. As used herein the disorder,disease, or abnormal state is an abnormal prostate state, includingbenign prostate hyperplasia and cancer, particularly prostate cancer.The abnormal prostate state of prostate cancer can be further subdividedinto stages and grades of prostate cancer as provided, for example inProstate. In: Edge S B, Byrd D R, Compton C C, et al., eds.: AJCC CancerStaging Manual. 7th ed. New York, N.Y.: Springer, 2010, pp 457-68(incorporated herein by reference in its entirety). Further, abnormalprostate states can be classified as one or more of benign prostatehyperplasia (BPH), androgen sensitive prostate cancer, androgeninsensitive or resistant prostate cancer, aggressive prostate cancer,non-aggressive prostate cancer, metastatic prostate cancer, andnon-metastatic prostate cancer.

As used herein, a sample obtained at an “earlier time point” is a samplethat was obtained at a sufficient time in the past such that clinicallyrelevant information could be obtained in the sample from the earliertime point as compared to the later time point. In certain embodiments,an earlier time point is at least four weeks earlier. In certainembodiments, an earlier time point is at least six weeks earlier. Incertain embodiments, an earlier time point is at least two monthsearlier. In certain embodiments, an earlier time point is at least threemonths earlier. In certain embodiments, an earlier time point is atleast six months earlier. In certain embodiments, an earlier time pointis at least nine months earlier. In certain embodiments, an earlier timepoint is at least one year earlier. Multiple subject samples (e.g., 3,4, 5, 6, 7, or more) can be obtained at regular or irregular intervalsover time and analyzed for trends in changes in marker levels.Appropriate intervals for testing for a particular subject can bedetermined by one of skill in the art based on ordinary considerations.

The term “expression” is used herein to mean the process by which apolypeptide is produced from DNA. The process involves the transcriptionof the gene into mRNA and the translation of this mRNA into apolypeptide. Depending on the context in which used, “expression” mayrefer to the production of RNA, or protein, or both.

As used herein, “greater predictive value” is understood as an assaythat has significantly greater sensitivity and/or specificity,preferably greater sensitivity and specificity, than the test to whichit is compared. The predictive value of a test can be determined usingan ROC analysis. In an ROC analysis a test that provides perfectdiscrimination or accuracy between normal and disease states would havean area under the curve (AUC)=1, whereas a very poor test that providesno better discrimination than random chance would have AUC=0.5. As usedherein, a test with a greater predictive value will have a statisticallyimproved AUC as compared to another assay. The assays are preformed inan appropriate subject population.

A “higher level of expression”, “higher level”, and the like of a markerrefers to an expression level in a test sample that is greater than thestandard error of the assay employed to assess expression, and ispreferably at least 25% more, at least 50% more, at least 75% more, atleast two, at least three, at least four, at least five, at least six,at least seven, at least eight, at least nine, or at least ten times theexpression level of the marker in a control sample (e.g., sample from ahealthy subject not having the marker associated disease, i.e., anabnormal prostate state) and preferably, the average expression level ofthe marker or markers in several control samples.

As used herein, the term “hybridization,” as in “nucleic acidhybridization,” refers generally to the hybridization of twosingle-stranded nucleic acid molecules having complementary basesequences, which under appropriate conditions will form athermodynamically favored double-stranded structure. Examples ofhybridization conditions can be found in the two laboratory manualsreferred above (Sambrook et al., 2000, supra and Ausubel et al., 1994,supra, or further in Higgins and Hames (Eds.) “Nucleic acidhybridization, a practical approach” IRL Press Oxford, Washington D.C.,(1985)) and are commonly known in the art. In the case of ahybridization to a nitrocellulose filter (or other such support likenylon), as for example in the well-known Southern blotting procedure, anitrocellulose filter can be incubated overnight at a temperaturerepresentative of the desired stringency condition (60-65° C. for highstringency, 50-60° C. for moderate stringency and 40-45° C. for lowstringency conditions) with a labeled probe in a solution containinghigh salt (6×SSC or 5×SSPE), 5×Denhardt's solution, 0.5% SDS, and 100μg/ml denatured carrier DNA (e.g., salmon sperm DNA). Thenon-specifically binding probe can then be washed off the filter byseveral washes in 0.2×SSC/0.1% SDS at a temperature which is selected inview of the desired stringency: room temperature (low stringency), 42°C. (moderate stringency) or 65° C. (high stringency). The salt and SDSconcentration of the washing solutions may also be adjusted toaccommodate for the desired stringency. The selected temperature andsalt concentration is based on the melting temperature (Tm) of the DNAhybrid. Of course, RNA-DNA hybrids can also be formed and detected. Insuch cases, the conditions of hybridization and washing can be adaptedaccording to well-known methods by the person of ordinary skillStringent conditions will be preferably used (Sambrook et al., 2000,supra). Other protocols or commercially available hybridization kits(e.g., ExpressHyb® from BD Biosciences Clonetech) using differentannealing and washing solutions can also be used as well known in theart. As is well known, the length of the probe and the composition ofthe nucleic acid to be determined constitute further parameters of thehybridization conditions. Note that variations in the above conditionsmay be accomplished through the inclusion and/or substitution ofalternate blocking reagents used to suppress background in hybridizationexperiments. Typical blocking reagents include Denhardt's reagent,BLOTTO, heparin, denatured salmon sperm DNA, and commercially availableproprietary formulations. The inclusion of specific blocking reagentsmay require modification of the hybridization conditions describedabove, due to problems with compatibility. Hybridizing nucleic acidmolecules also comprise fragments of the above described molecules.Furthermore, nucleic acid molecules which hybridize with any of theaforementioned nucleic acid molecules also include complementaryfragments, derivatives and allelic variants of these molecules.Additionally, a hybridization complex refers to a complex between twonucleic acid sequences by virtue of the formation of hydrogen bondsbetween complementary G and C bases and between complementary A and Tbases; these hydrogen bonds may be further stabilized by base stackinginteractions. The two complementary nucleic acid sequences hydrogen bondin an antiparallel configuration. A hybridization complex may be formedin solution (e.g., Cot or Rot analysis) or between one nucleic acidsequence present in solution and another nucleic acid sequenceimmobilized on a solid support (e.g., membranes, filters, chips, pins orglass slides to which, e.g., cells have been fixed).

As used herein, the term “identical” or “percent identity” in thecontext of two or more nucleic acid or amino acid sequences, refers totwo or more sequences or subsequences that are the same, or that have aspecified percentage of amino acid residues or nucleotides that are thesame (e.g., 60% or 65% identity, preferably, 70-95% identity, morepreferably at least 95% identity), when compared and aligned for maximumcorrespondence over a window of comparison, or over a designated regionas measured using a sequence comparison algorithm as known in the art,or by manual alignment and visual inspection. Sequences having, forexample, 60% to 95% or greater sequence identity are considered to besubstantially identical. Such a definition also applies to thecomplement of a test sequence. Preferably the described identity existsover a region that is at least about 15 to 25 amino acids or nucleotidesin length, more preferably, over a region that is about 50 to 100 aminoacids or nucleotides in length. Those having skill in the art will knowhow to determine percent identity between/among sequences using, forexample, algorithms such as those based on CLUSTALW computer program(Thompson Nucl. Acids Res. 2 (1994), 4673-4680) or FASTDB (Brutlag Comp.App. Biosci. 6 (1990), 237-245), as known in the art. Although theFASTDB algorithm typically does not consider internal non-matchingdeletions or additions in sequences, i.e., gaps, in its calculation,this can be corrected manually to avoid an overestimation of the %identity. CLUSTALW, however, does take sequence gaps into account in itsidentity calculations. Also available to those having skill in this artare the BLAST and BLAST 2.0 algorithms (Altschul Nucl. Acids Res. 25(1977), 3389-3402). The BLASTN program for nucleic acid sequences usesas defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4,and a comparison of both strands. For amino acid sequences, the BLASTPprogram uses as defaults a wordlength (W) of 3, and an expectation (E)of 10. The BLOSUM62 scoring matrix (Henikoff Proc. Natl. Acad. Sci.,USA, 89, (1989), 10915) uses alignments (B) of 50, expectation (E) of10, M=5, N=4, and a comparison of both strands. Moreover, the presentinvention also relates to nucleic acid molecules the sequence of whichis degenerate in comparison with the sequence of an above-describedhybridizing molecule. When used in accordance with the present inventionthe term “being degenerate as a result of the genetic code” means thatdue to the redundancy of the genetic code different nucleotide sequencescode for the same amino acid. The present invention also relates tonucleic acid molecules which comprise one or more mutations ordeletions, and to nucleic acid molecules which hybridize to one of theherein described nucleic acid molecules, which show (a) mutation(s) or(a) deletion(s).

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to.”

A subject at “increased risk for developing prostate cancer” may or maynot develop prostate cancer. Identification of a subject at increasedrisk for developing prostate cancer should be monitored for additionalsigns or symptoms of prostate cancer. The methods provided herein foridentifying a subject with increased risk for developing prostate cancercan be used in combination with assessment of other known risk factorsor signs of prostate cancer including, but not limited to decreasedurinary stream, urgency, hesitancy, nocturia, incomplete bladderemptying, and age.

As used herein, the term “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments can consist of, but are not limitedto, test tubes and cell culture. The term “in vivo” refers to thenatural environment (e.g., an animal or a cell) and to processes orreaction that occur within a natural environment.

As used herein, a “label” refers to a molecular moiety or compound thatcan be detected or can lead to a detectable signal. A label is joined,directly or indirectly, to a molecule, such as an antibody, a nucleicacid probe or the protein/antigen or nucleic acid to be detected (e.g.,an amplified sequence). Direct labeling can occur through bonds orinteractions that link the label to the nucleic acid (e.g., covalentbonds or non-covalent interactions), whereas indirect labeling can occurthrough the use of a “linker” or bridging moiety, such asoligonucleotide(s) or small molecule carbon chains, which is eitherdirectly or indirectly labeled. Bridging moieties may amplify adetectable signal. Labels can include any detectable moiety (e.g., aradionuclide, ligand such as biotin or avidin, enzyme or enzymesubstrate, reactive group, chromophore such as a dye or coloredparticle, luminescent compound including a bioluminescent,phosphorescent or chemiluminescent compound, and fluorescent compound).Preferably, the label on a labeled probe is detectable in a homogeneousassay system, i.e., in a mixture, the bound label exhibits a detectablechange compared to an unbound label.

The terms “level of expression of a gene”, “gene expression level”,“level of a marker”, and the like refer to the level of mRNA, as well aspre-mRNA nascent transcript(s), transcript processing intermediates,mature mRNA(s) and degradation products, or the level of protein,encoded by the gene in the cell. The “level” of one of more biomarkersmeans the absolute or relative amount or concentration of the biomarkerin the sample.

A “lower level of expression” or “lower level” of a marker refers to anexpression level in a test sample that is less than 90%, 85%, 80%, 75%,70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% ofthe expression level of the marker in a control sample (e.g., samplefrom a healthy subjects not having the marker associated disease, i.e.,an abnormal prostate state) and preferably, the average expression levelof the marker in several control samples.

The term “modulation” refers to upregulation (i.e., activation orstimulation), down-regulation (i.e., inhibition or suppression) of aresponse (e.g., level of expression of a marker), or the two incombination or apart. A “modulator” is a compound or molecule thatmodulates, and may be, e.g., an agonist, antagonist, activator,stimulator, suppressor, or inhibitor.

As used herein, “negative fold change” refers to “down-regulation” or“decrease (of expression)” of a gene that is listed herein.

As used herein, “nucleic acid molecule” or “polynucleotides”, refers toa polymer of nucleotides. Non-limiting examples thereof include DNA(e.g., genomic DNA, cDNA), RNA molecules (e.g., mRNA) and chimerasthereof. The nucleic acid molecule can be obtained by cloning techniquesor synthesized. DNA can be double-stranded or single-stranded (codingstrand or non-coding strand [antisense]). Conventional ribonucleic acid(RNA) and deoxyribonucleic acid (DNA) are included in the term “nucleicacid” and polynucleotides as are analogs thereof. A nucleic acidbackbone may comprise a variety of linkages known in the art, includingone or more of sugar-phosphodiester linkages, peptide-nucleic acid bonds(referred to as “peptide nucleic acids” (PNA); Hydig-Hielsen et al., PCTIntl Pub. No. WO 95/32305), phosphorothioate linkages, methylphosphonatelinkages or combinations thereof. Sugar moieties of the nucleic acid maybe ribose or deoxyribose, or similar compounds having knownsubstitutions, e.g., 2′ methoxy substitutions (containing a2′-O-methylribofuranosyl moiety; see PCT No. WO 98/02582) and/or 2′halide substitutions. Nitrogenous bases may be conventional bases (A, G,C, T, U), known analogs thereof (e.g., inosine or others; see TheBiochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11th ed.,1992), or known derivatives of purine or pyrimidine bases (see, Cook,PCT Int'l Pub. No. WO 93/13121) or “abasic” residues in which thebackbone includes no nitrogenous base for one or more residues (Arnoldet al., U.S. Pat. No. 5,585,481). A nucleic acid may comprise onlyconventional sugars, bases and linkages, as found in RNA and DNA, or mayinclude both conventional components and substitutions (e.g.,conventional bases linked via a methoxy backbone, or a nucleic acidincluding conventional bases and one or more base analogs). An “isolatednucleic acid molecule”, as is generally understood and used herein,refers to a polymer of nucleotides, and includes, but should not limitedto DNA and RNA. The “isolated” nucleic acid molecule is purified fromits natural in vivo state, obtained by cloning or chemicallysynthesized.

As used herein, the term “obtaining” is understood herein asmanufacturing, purchasing, or otherwise coming into possession of.

As used herein, “oligonucleotides” or “oligos” define a molecule havingtwo or more nucleotides (ribo or deoxyribonucleotides). The size of theoligo will be dictated by the particular situation and ultimately on theparticular use thereof and adapted accordingly by the person of ordinaryskill. An oligonucleotide can be synthesized chemically or derived bycloning according to well-known methods. While they are usually in asingle-stranded form, they can be in a double-stranded form and evencontain a “regulatory region”. They can contain natural rare orsynthetic nucleotides. They can be designed to enhance a chosen criterialike stability for example. Chimeras of deoxyribonucleotides andribonucleotides may also be within the scope of the present invention.

As used herein, “one or more” is understood as each value 1, 2, 3, 4, 5,6, 7, 8, 9, 10, and any value greater than 10.

The term “or” is used inclusively herein to mean, and is usedinterchangeably with, the term “and/or,” unless context clearlyindicates otherwise. For example, as used herein, filamin B or LY9 isunderstood to include filamin B alone, LY9 alone, and the combination offilamin B and LY9.

As used herein, “patient” or “subject” can mean either a human ornon-human animal, preferably a mammal. By “subject” is meant any animal,including horses, dogs, cats, pigs, goats, rabbits, hamsters, monkeys,guinea pigs, rats, mice, lizards, snakes, sheep, cattle, fish, andbirds. A human subject may be referred to as a patient. It should benoted that clinical observations described herein were made with humansubjects and, in at least some embodiments, the subjects are human.

As used herein, “positive fold change” refers to “up-regulation” or“increase (of expression)” of a gene that is listed herein.

As used herein, “preventing” or “prevention” refers to a reduction inrisk of acquiring a disease or disorder (i.e., causing at least one ofthe clinical symptoms of the disease not to develop in a patient thatmay be exposed to or predisposed to the disease but does not yetexperience or display symptoms of the disease). Prevention does notrequire that the disease or condition never occurs in the subject.Prevention includes delaying the onset or severity of the disease orcondition.

As used herein, a “predetermined threshold value” or “threshold value”of a biomarker refers to the level of the biomarker (e.g., theexpression level or quantity (e.g., ng/ml) in a biological sample) in acorresponding control/normal sample or group of control/normal samplesobtained from normal or healthy subjects, e.g., those males that do nothave prostate cancer. The predetermined threshold value may bedetermined prior to or concurrently with measurement of marker levels ina biological sample. The control sample may be from the same subject ata previous time or from different subjects.

As used herein, a “probe” is meant to include a nucleic acid oligomer oroligonucleotide that hybridizes specifically to a target sequence in anucleic acid or its complement, under conditions that promotehybridization, thereby allowing detection of the target sequence or itsamplified nucleic acid. Detection may either be direct (i.e., resultingfrom a probe hybridizing directly to the target or amplified sequence)or indirect (i.e., resulting from a probe hybridizing to an intermediatemolecular structure that links the probe to the target or amplifiedsequence). A probe's “target” generally refers to a sequence within anamplified nucleic acid sequence (i.e., a subset of the amplifiedsequence) that hybridizes specifically to at least a portion of theprobe sequence by standard hydrogen bonding or “base pairing.” Sequencesthat are “sufficiently complementary” allow stable hybridization of aprobe sequence to a target sequence, even if the two sequences are notcompletely complementary. A probe may be labeled or unlabeled. A probecan be produced by molecular cloning of a specific DNA sequence or itcan also be synthesized. Numerous primers and probes which can bedesigned and used in the context of the present invention can be readilydetermined by a person of ordinary skill in the art to which the presentinvention pertains.

As used herein, the terminology “prognosis”, “staging” and“determination of aggressiveness” are defined herein as the predictionof the degree of severity of the prostate cancer and of its evolution aswell as the prospect of recovery as anticipated from usual course of thedisease. According to the present invention, once the aggressiveness ofthe prostate cancer has been determined appropriate methods oftreatments can be chosen.

As used herein, “prophylactic” or “therapeutic” treatment refers toadministration to the subject of one or more agents or interventions toprovide the desired clinical effect. If it is administered prior toclinical manifestation of the unwanted condition (e.g., disease or otherunwanted state of the host animal) then the treatment is prophylactic,i.e., it protects the host against developing at least one sign orsymptom of the unwanted condition, whereas if administered aftermanifestation of the unwanted condition, the treatment is therapeutic(i.e., it is intended to diminish, ameliorate, or maintain at least onesign or symptom of the existing unwanted condition or side effectstherefrom).

As used herein, “prostate cancer,” refers to any malignant orpre-malignant form of cancer of the prostate. The term includes prostatein situ carcinomas, invasive carcinomas, metastatic carcimomas andpre-malignant conditions. The term also encompasses any stage or gradeof cancer in the prostate. Where the prostate cancer is “metastatic,”the cancer has spread or metastasized beyond the prostate gland to adistant site, such as a lymph node or to the bone.

As used herein, a “reference level” of a biomarker means a level of thebiomarker that is indicative of a particular disease state, phenotype,or lack thereof, as well as combinations of disease states, phenotypes,or lack thereof. A “positive” reference level of a biomarker means alevel that is indicative of a particular disease state or phenotype. A“negative” reference level of a biomarker means a level that isindicative of a lack of a particular disease state or phenotype. Forexample, a “prostate cancer-positive reference level” of a biomarkermeans a level of a biomarker that is indicative of a positive diagnosisof prostate cancer in a subject, and a “prostate cancer-negativereference level” of a biomarker means a level of a biomarker that isindicative of a negative diagnosis of prostate cancer in a subject. A“reference level” of a biomarker may be an absolute or relative amountor concentration of the biomarker, a presence or absence of thebiomarker, a range of amount or concentration of the biomarker, aminimum and/or maximum amount or concentration of the biomarker, a meanamount or concentration of the biomarker, and/or a median amount orconcentration of the biomarker; and, in addition, “reference levels” ofcombinations of biomarkers may also be ratios of absolute or relativeamounts or concentrations of two or more biomarkers with respect to eachother. Appropriate positive and negative reference levels of biomarkersfor a particular disease state, phenotype, or lack thereof may bedetermined by measuring levels of desired biomarkers in one or moreappropriate subjects, and such reference levels may be tailored tospecific populations of subjects (e.g., a reference level may beage-matched so that comparisons may be made between biomarker levels insamples from subjects of a certain age and reference levels for aparticular disease state, phenotype, or lack thereof in a certain agegroup). Such reference levels may also be tailored to specifictechniques that are used to measure levels of biomarkers in biologicalsamples (e.g., LC-MS, GC-MS, etc.), where the levels of biomarkers maydiffer based on the specific technique that is used.

As used herein, “sample” or “biological sample” includes a specimen orculture obtained from any source. Biological samples can be obtainedfrom blood (including any blood product, such as whole blood, plasma,serum, or specific types of cells of the blood), urine, saliva, and thelike. Biological samples also include tissue samples, such as biopsytissues or pathological tissues that have previously been fixed (e.g.,formaline snap frozen, cytological processing, etc.). In an embodiment,the biological sample is from blood. In another embodiment, thebiological sample is a biopsy tissue from the prostate gland.

As use herein, the phrase “specific binding” or “specifically binding”when used in reference to the interaction of an antibody and a proteinor peptide means that the interaction is dependent upon the presence ofa particular structure (i.e., the antigenic determinant or epitope) onthe protein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A,” the presence of aprotein containing epitope A (or free, unlabeled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

The phrase “specific identification” is understood as detection of amarker of interest with sufficiently low background of the assay andcross-reactivity of the reagents used such that the detection method isdiagnostically useful. In certain embodiments, reagents for specificidentification of a marker bind to only one isoform of the marker. Incertain embodiments, reagents for specific identification of a markerbind to more than one isoform of the marker. In certain embodiments,reagents for specific identification of a marker bind to all knownisoforms of the marker.

As used herein, the phrase “subject suspected of having cancer” refersto a subject that presents one or more symptoms indicative of a canceror is being screened for a cancer (e.g., during a routine physical). Asubject suspected of having cancer may also have one or more riskfactors. A subject suspected of having cancer has generally not beentested for cancer. However, a “subject suspected of having cancer”encompasses an individual who has received an initial diagnosis (e.g., aCT scan showing a mass or increased PSA level) but for whom the stage ofcancer is not known. The term further includes people who once hadcancer (e.g., an individual in remission).

The term “such as” is used herein to mean, and is used interchangeably,with the phrase “such as but not limited to.”

As used herein, the term “stage of cancer” refers to a qualitative orquantitative assessment of the level of advancement of a cancer.Criteria used to determine the stage of a cancer include, but are notlimited to, the size of the tumor, whether the tumor has spread to otherparts of the body and where the cancer has spread (e.g., within the sameorgan or region of the body or to another organ).

As used herein, the term “staging” refers to commonly used systems forgrading/stating cancer, e.g., prostate cancer. In one aspect, stagingcan take the form of the “Gleason Score”, as well known in the art, isthe most commonly used system for the grading/staging and prognosis ofadenocarcinoma. The system describes a score between 2 and 10, with 2being the least aggressive and 10 being the most aggressive. The scoreis the sum of the two most common patterns (grade 1-5) of tumor growthfound. To be counted a pattern (grade) needs to occupy more than 5% ofthe biopsy specimen. The scoring system requires biopsy material (corebiopsy or operative specimens) in order to be accurate; cytologicalpreparations cannot be used. The “Gleason Grade” is the most commonlyused prostate cancer grading system. It involves assigning numbers tocancerous prostate tissue, ranging from 1 through 5, based on how muchthe arrangement of the cancer cells mimics the way normal prostate cellsform glands. Two grades are assigned to the most common patterns ofcells that appear; these two grades (they can be the same or different)are then added together to determine the Gleason score (a number from 1to 10). The Gleason system is based exclusively on the architecturalpattern of the glands of the prostate tumor. It evaluates howeffectively the cells of any particular cancer are able to structurethemselves into glands resembling those of the normal prostate. Theability of a tumor to mimic normal gland architecture is called itsdifferentiation, and experience has shown that a tumor whose structureis nearly normal (well differentiated) will probably have a biologicalbehavior relatively close to normal, i.e., that is not very aggressivelymalignant.

A Gleason grading from very well differentiated (grade 1) to very poorlydifferentiated (grade 5) is usually done for the most part by viewingthe low magnification microscopic image of the cancer. There areimportant additional details which require higher magnification, and anability to accurately grade any tumor is achieved only through muchtraining and experience in pathology. Gleason grades 1 and 2: These twogrades closely resemble normal prostate. They are the least importantgrades because they seldom occur in the general population and becausethey confer a prognostic benefit which is only slightly better thangrade 3. Both of these grades are composed by mass; in grade 2 they aremore loosely aggregated, and some glands wander (invade) into thesurrounding muscle (stroma). Gleason grade 3 is the most common gradeand is also considered well differentiated (like grades 1 and 2). Thisis because all three grades have a normal “gland unit” like that of anormal prostate; that is, every cell is part of a circular row whichforms the lining of a central space (the lumen). The lumen containsprostatic secretion like normal prostate, and each gland unit issurrounded by prostate muscle which keeps the gland units apart. Incontrast to grade 2, wandering of glands (invading) into the stroma(muscle) is very prominent and is the main defining feature. The cellsare dark rather than pale and the glands often have more variableshapes.

Gleason Grade 4 is probably the most important grade because it isfairly common and because of the fact that if a lot of it is present,patient prognosis is usually (but not always) worsened by a considerabledegree. Grade 4 also shows a considerable loss of architecture. For thefirst time, disruption and loss of the normal gland unit is observed. Infact, grade 4 is identified almost entirely by loss of the ability toform individual, separate gland units, each with its separate lumen(secretory space). This important distinction is simple in concept butcomplex in practice. The reason is that there are a variety ofdifferent-appearing ways in which the cancer's effort to form glandunits can be distorted. Each cancer has its own partial set of toolswith which it builds part of the normal structure. Grade 4 is like thebranches of a large tree, reaching in a number of directions from the(well differentiated) trunk of grades 1, 2, and 3. Much experience isrequired for this diagnosis, and not all patterns are easilydistinguished from grade 3. This is the main class of poorlydifferentiated prostate cancer, and its distinction from grade 3 is themost commonly important grading decision.

Gleason grade 5 is an important grade because it usually predictsanother significant step towards poor prognosis. Its overall importancefor the general population is reduced by the fact that it is less commonthan grade 4, and it is seldom seen in men whose prostate cancer isdiagnosed early in its development. This grade too shows a variety ofpatterns, all of which demonstrate no evidence of any attempt to formgland units. This grade is often called undifferentiated, because itsfeatures are not significantly distinguishing to make it look anydifferent from undifferentiated cancers which occur in other organs.When a pathologist looks at prostate cancer specimens under themicroscope and gives them a Gleason grade, an attempt to identify twoarchitectural patterns and assign a Gleason grade to each one is made.There may be a primary or most common pattern and then a secondary orsecond most common pattern which the pathologist will seek to describefor each specimen; alternatively, there may often be only a single puregrade. In developing his system, Dr. Gleason discovered that by giving acombination of the grades of the two most common patterns he could seein any particular patient's specimens, that he was better able topredict the likelihood that a particular patient would do well or badly.Therefore, although it may seem confusing, the Gleason score which aphysician usually gives to a patient, is actually a combination or sumof two numbers which is accurate enough to be very widely used. Thesecombined Gleason sums or scores may be determined as follows:

The lowest possible Gleason score is 2 (1+1), where both the primary andsecondary patterns have a Gleason grade of 1 and therefore when addedtogether their combined sum is 2.

Very typical Gleason scores might be 5 (2+3), where the primary patternhas a Gleason grade of 2 and the secondary pattern has a grade of 3, or6 (3+3), a pure pattern.

Another typical Gleason score might be 7 (4+3), where the primarypattern has a Gleason grade of 4 and the secondary pattern has a gradeof 3.

Finally, the highest possible Gleason score is 10 (5+5), when theprimary and secondary patterns both have the most disordered Gleasongrades of 5.

Another way of staging prostate cancer is by using the TNM System. Itdescribes the extent of the primary tumor (T stage), the absence orpresence of spread to nearby lymph nodes (N stage) and the absence orpresence of distant spread, or metastasis (M stage). Each category ofthe TNM classification is divided into subcategories representative ofits particular state. For example, primary tumors (T stage) may beclassified into:

T1: The tumor cannot be felt during a digital rectal exam, or seen byimaging studies, but cancer cells are found in a biopsy specimen;

T2: The tumor can be felt during a DRE and the cancer is confined withinthe prostate gland;

T3: The tumor has extended through the prostatic capsule (a layer offibrous tissue surrounding the prostate gland) and/or to the seminalvesicles (two small sacs next to the prostate that store semen), but noother organs are affected;

T4: The tumor has spread or attached to tissues next to the prostate(other than the seminal vesicles).

Lymph node involvement is divided into the following 4 categories:

N0: Cancer has not spread to any lymph nodes;

N1: Cancer has spread to a single regional lymph node (inside thepelvis) and is not larger than 2 centimeters;

N2: Cancer has spread to one or more regional lymph nodes and is largerthan 2 centimeters, but not larger than 5 centimeters; and

N3: Cancer has spread to a lymph node and is larger than 5 centimeters(2 inches).

Metastasis is generally divided into the following two categories:

M0: The cancer has not metastasized (spread) beyond the regional lymphnodes; and

M1: The cancer has metastasized to distant lymph nodes (outside of thepelvis), bones, or other distant organs such as lungs, liver, or brain.

In addition, the Tstage is further divided into subcategories T1a-cT2a-c, T3a-c and T4a-b. The characteristics of each of thesesubcategories are well known in the art and can be found in a number oftextbooks.

The terms “test compound” and “candidate compound” refer to any chemicalentity, pharmaceutical, drug, and the like that is a candidate for useto treat or prevent a disease, illness, sickness, or disorder of bodilyfunction (e.g., cancer). Test compounds comprise both known andpotential therapeutic compounds. A test compound can be determined to betherapeutic by screening using the screening methods of the presentinvention. In some embodiments of the present invention, test compoundsinclude antisense compounds.

The term “therapeutic effect” refers to a local or systemic effect inanimals, particularly mammals, and more particularly humans caused by apharmacologically active substance. The term thus means any substanceintended for use in the diagnosis, cure, mitigation, treatment, orprevention of disease, or in the enhancement of desirable physical ormental development and conditions in an animal or human A therapeuticeffect can be understood as a decrease in tumor growth, decrease intumor growth rate, stabilization or decrease in tumor burden,stabilization or reduction in tumor size, stabilization or decrease intumor malignancy, increase in tumor apoptosis, and/or a decrease intumor angiogenesis.

As used herein, “therapeutically effective amount” means the amount of acompound that, when administered to a patient for treating a disease, issufficient to effect such treatment for the disease, e.g., the amount ofsuch a substance that produces some desired local or systemic effect ata reasonable benefit/risk ratio applicable to any treatment, e.g., issufficient to ameliorate at least one sign or symptom of the disease,e.g., to prevent progression of the disease or condition, e.g., preventtumor growth, decrease tumor size, induce tumor cell apoptosis, reducetumor angiogenesis, prevent metastasis. When administered for preventinga disease, the amount is sufficient to avoid or delay onset of thedisease. The “therapeutically effective amount” will vary depending onthe compound, its therapeutic index, solubility, the disease and itsseverity and the age, weight, etc., of the patient to be treated, andthe like. For example, certain compounds discovered by the methods ofthe present invention may be administered in a sufficient amount toproduce a reasonable benefit/risk ratio applicable to such treatment.Administration of a therapeutically effective amount of a compound mayrequire the administration of more than one dose of the compound.

A “transcribed polynucleotide” or “nucleotide transcript” is apolynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such RNA orcDNA) which is complementary to or having a high percentage of identity(e.g., at least 80% identity) with all or a portion of a mature mRNAmade by transcription of a marker of the invention and normalpost-transcriptional processing (e.g. splicing), if any, of the RNAtranscript, and reverse transcription of the RNA transcript.

As used herein, “treatment,” particularly “active treatment,” refers toperforming an intervention to treat prostate cancer in a subject, e.g.,reduce at least one of the growth rate, reduction of tumor burden,reduce or maintain the tumor size, or the malignancy (e.g., likelihoodof metastasis) of the tumor; or to increase apoptosis in the tumor byone or more of administration of a therapeutic agent, e.g., chemotherapyor hormone therapy; administration of radiation therapy (e.g., pelletimplantation, brachytherapy), or surgical resection of the tumor, or anycombination thereof appropriate for treatment of the subject based ongrade and stage of the tumor and other routine considerations. Activetreatment is distinguished from “watchful waiting” (i.e., not activetreatment) in which the subject and tumor are monitored, but nointerventions are performed to affect the tumor. Watchful waiting caninclude administration of agents that alter effects caused by the tumor(e.g., incontinence, erectile dysfunction) that are not administered toalter the growth or pathology of the tumor itself.

The recitation of a listing of chemical group(s) in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Reference will now be made in detail to exemplary embodiments of theinvention. While the invention will be described in conjunction with theexemplary embodiments, it will be understood that it is not intended tolimit the invention to those embodiments. To the contrary, it isintended to cover alternatives, modifications, and equivalents as may beincluded within the spirit and scope of the invention as defined by theappended claims.

Exemplary compositions and methods of the present invention aredescribed in more detail in the following sections: (C) Biomarkers ofthe invention; (D) Prostate tissue samples; (E) Detection and/ormeasurement of the biomarkers of the invention; (F) Isolated biomarkers;(G) Applications of biomarkers of the invention; (H) Therapeutics; (I)Drug screening; and (J) Kits/panels.

C. Biomarkers of the Invention

The present invention is based, at least in part, on the discovery thatfilamin A is differentially regulated in prostate cancer cells. Inparticular, the invention is based on the surprising discovery thatfilamin A levels are significantly elevated in the serum of patientswith prostate cancer. Accordingly, the invention provides methods fordiagnosing and/or monitoring (e.g., monitoring of disease progression ortreatment) and/or prognosing an oncological disease state, e.g.,prostate cancer, in a mammal. The invention also provides methods fortreating or for adjusting treatment regimens based on diagnosticinformation relating to the levels of filamin A in the serum of asubject with an oncological disease state, e.g., prostate cancer. Theinvention further provides panels and kits for practicing the methods ofthe invention.

The present invention provides new biomarkers and combinations ofbiomarkers for use in diagnosing and/or prognosing an oncologicaldisorder, and in particular, biomarkers for use in diagnosing and/orprognosing prostate cancer. The biomarkers of the invention are meant toencompass any measurable characteristic that reflects in a quantitativeor qualitative manner the physiological state of an organism, e.g,whether the organism has prostate cancer. The physiological state of anorganism is inclusive of any disease or non-disease state, e.g., asubject having prostate cancer or a subject who is otherwise healthy.Said another way, the biomarkers of the invention includecharacteristics that can be objectively measured and evaluated asindicators of normal processes, pathogenic processes, or pharmacologicresponses to a therapeutic intervention, including, in particular,prostate cancer. Biomarkers can be clinical parameters (e.g., age,performance status), laboratory measures (e.g., molecular biomarkers,such as prostate specific antigen), imaging-based measures, or geneticor other molecular determinants, as well as combinations thereof.Examples of biomarkers include, for example, polypeptides, peptides,polypeptide fragments, proteins, antibodies, hormones, polynucleotides,RNA or RNA fragments, microRNA (miRNAs), lipids, polysaccharides, andother bodily metabolites that are diagnostic and/or indicative and/orpredictive of an oncological disease, e.g., prostate cancer. Examples ofbiomarkers also include polypeptides, peptides, polypeptide fragments,proteins, antibodies, hormones, polynucleotides, RNA or RNA fragments,microRNA (miRNAs), lipids, polysaccharides, and other bodily metaboliteswhich are diagnostic and/or indicative and/or predictive of any stage orclinical phase of an oncological disease, such as, prostate cancer.Clinical stage or phase can be represented by any means known in theart, for example, based on the Gleason Score system, e.g., Gleason grade1, grade 2, grade 3, grade 4, or grade 5 prostate cancer.

In one aspect, the present invention relates to using, measuring,detecting, and the like of filamin A alone, or together with one or moreadditional biomarkers of prostate cancer, which can include, but are notlimited to prostate specific antigen (PSA), filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1. Other markers thatmay be used in combination with filamin A include any measurablecharacteristic described herein that reflects in a quantitative orqualitative manner the physiological state of an organism, e.g, whetherthe organism has prostate cancer. The physiological state of an organismis inclusive of any disease or non-disease state, e.g., a subject havingprostate cancer or a subject who is otherwise healthy. The biomarkers ofthe invention that may be used in combination with filamin A includecharacteristics that can be objectively measured and evaluated asindicators of normal processes, pathogenic processes, or pharmacologicresponses to a therapeutic intervention, including, in particular,prostate cancer. Such combination biomarkers can be clinical parameters(e.g., age, performance status), laboratory measures (e.g., molecularbiomarkers, such as prostate specific antigen), imaging-based measures,or genetic or other molecular determinants Examples of biomarkers foruse in combination with filamin A include, for example, polypeptides,peptides, polypeptide fragments, proteins, antibodies, hormones,polynucleotides, RNA or RNA fragments, microRNA (miRNAs), lipids,polysaccharides, and other bodily metabolites that are diagnostic and/orindicative and/or predictive of prostate cancer, or any particular stageor phase of prostate cancer, e.g., Gleason grade 1, grade 2, grade 3,grade 4, or grade 5 prostate cancer or TNM classifications. In otherembodiments, the present invention also involves the analysis andconsideration of any clinical and/or patient-related health data, forexample, data obtained from an Electronic Medical Record (e.g.,collection of electronic health information about individual patients orpopulations relating to various types of data, such as, demographics,medical history, medication and allergies, immunization status,laboratory test results, radiology images, vital signs, personalstatistics like age and weight, and billing information).

In certain embodiments, filamin A may be used in combination with atleast one other biomarker, or more preferably, with at least two otherbiomarkers, or still more preferably, with at least three otherbiomarkers, or even more preferably with at least four other biomarkers.Still further, filamin A in certain embodiments, may be used incombination with at least five other markers, or at least six otherbiomarkers, or at least seven other biomarkers, or at least eight otherbiomarkers, or at least nine other biomarkers, or at least ten otherbiomarkers, or at least eleven other biomarkers, or at least twelveother biomarkers, or at least thirteen other biomarkers, or at leastfourteen other biomarkers, or at least fifteen other biomarkers, or atleast sixteen other biomarkers, or at least seventeen other biomarkers,or at least eighteen other biomarkers, or at least nineteen otherbiomarkers, or at least twenty other biomarkers. Further, filamin A maybe used in combination with a multitude of other biomarkers, including,for example, with between about 20-50 other biomarkers, or between50-100, or between 100-500, or between 500-1000, or between 1000-10,000or biomarkers or more.

In certain embodiments, the present invention involves the detectionand/or analysis filamin A I combination with at least one of thefollowing set of biomarkers: prostate specific antigen (PSA), filamin B,LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1.The known function of these proteins is provided as follows, withoutwishing to be bound by theory:

Filamin A (FLN-A). Filamin A (also known as FLN-A, FLN1, ABP-280, OPD1,OPD2, Endothelial Actin-Binding Protein, CVD1, FMD, MNS, NHBP, XLVD,XMVD, Actin Binding Protein 280, Alpha-Filamin, Filamin-1,Filamin-A—each of which may appear herein and are considered equivalentterms as used herein) is a 280-kD protein that is thought to crosslinkactin filaments into orthogonal networks in cortical cytoplasm. Thelarge molecular-weight protein also participates in the anchoring ofmembrane proteins to the actin cytoskeleton. Remodeling of thecytoskeleton is central to the modulation of cell shape and migrationcells. Filamin A, encoded by the FLNA gene, is a widely expressedprotein that regulates reorganization of the actin cytoskeleton byinteracting with integrins, transmembrane receptor complexes, and secondmessengers. At least two different isoforms are know, isoform 1 andisoform 2, all of which are contemplated by the invention andencompassed by the term “filamin A.” It will be appreciated that isoform1 is the predominant transcript encoding filamin A. Isoform 2 includesan alternate in-frame exon and encodes a slightly longer proteinisoform. Interaction with FLNA may allow neuroblast migration from theventricular zone into the cortical plate. FLNA tethers cellsurface-localized furin, modulates its rate of internalization anddirects its intracellular trafficking. Further reference to filamin Acan be found in the scientific literature, for example, in Gorlin J B etla., (October 1993). “Actin-binding protein (ABP-280) filamin gene (FLN)maps telomeric to the color vision locus (R/GCP) and centromeric to G6PDin Xq28”. Genomics 17 (2): 496-8, and Robertson S P et al. (March 2003).“Localized mutations in the gene encoding the cytoskeletal proteinfilamin A cause diverse malformations in humans”. Nat Genet 33 (4):487-91, each of which are incorporated herein by reference. Thenucleotide and amino acid sequences of filamin A can be found as GenBankAccession No. NM_001456.3 (filamin A—isoform 1—mRNA transcriptsequence—SEQ ID NO: 46) and the corresponding polypeptide sequence ofNP_001447.2 (filamin A—isoform 1—polypeptide sequence—SEQ ID NO: 47) andas GenBank Accession No. NM_001110556.1 (filamin A—isoform 2—mRNAtranscript sequence—SEQ ID NO: 48) and the corresponding polypeptidesequence of NP_001104026.1 (filamin A—isoform 2—polypeptide sequence—SEQID NO: 49). These GenBank numbers are incorporated herein by referencein the versions available on the earliest effective filing date of thisapplication.

The present invention is based, at least in part, on the discovery thatfilamin A is differentially regulated in prostate cancer cells. Inparticular, the invention is based on the surprising discovery thatfilamin A levels are significantly elevated in the serum of patientswith prostate cancer. Accordingly, the invention provides methods fordiagnosing and/or monitoring (e.g., monitoring of disease progression ortreatment) and/or prognosing an oncological disease state, e.g.,prostate cancer, in a mammal. The invention also provides methods fortreating or for adjusting treatment regimens based on diagnosticinformation relating to the levels of filamin A in the serum of asubject with an oncological disease state, e.g., prostate cancer. Theinvention further provides panels and kits for practicing the methods ofthe invention.

It is understood that the invention includes the use of any combinationof one or more of the filamin A sequences provided in the sequencelisting or any fragments thereof as long as the fragment can allow forthe specific identification of filamin A. Methods of the invention andreagents can be used to detect single isoforms of filamin A, e.g.,isoform 1 and isoform 2, combinations of filamin A isoforms, or all ofthe filamin A isoforms simultaneously. Unless specified, filamin A canbe considered to refer to one or more isoforms of filamin A, includingtotal filamin A. Moreover, it is understood that there are naturallyoccurring variants of filamin A, which may or may not be associated witha specific disease state, the use of which are also included in theinstant application.

It is understood that the invention includes the use of any fragments offilamin A polypeptide as long as the fragment allows for the specificidentification of filamin A by a detection method of the invention. Forexample, an ELISA antibody must be able to bind to the filamin Afragment so that detection is possible. Moreover, it is understood thatthere are naturally occurring variants of filamin A which may or may notbe associated with a specific disease state, the use of which are alsoincluded in this application. Accordingly, the present inventions alsocontemplates fragments and variants of filamin A which may be associatedwith a disease state, e.g., prostate cancer, and/or a particular stageor phase of a disease state, e.g., grades 1-5 of prostate cancer. It isalso understood that the invention encompasses the use of nucleic acidmolecules encoding filamin A, including for example, filamin A-encodingDNA, filamin A mRNA, and fragments and/or variants thereof. Reference to“filamin A” may refer to filamin A polypeptide or to the filamin A gene,unless otherwise indicated.

Keratin 4.

Keratin 4, also known as as K4; CK4; CK-4; CYK4, is a member of thekeratin gene family. The type II cytokeratins consist of basic orneutral proteins which are arranged in pairs of heterotypic keratinchains coexpressed during differentiation of simple and stratifiedepithelial tissues. This type II cytokeratin is specifically expressedin differentiated layers of the mucosal and esophageal epithelia withfamily member KRT13. Mutations in these genes have been associated withWhite Sponge Nevus, characterized by oral, esophageal, and analleukoplakia. The type II cytokeratins are clustered in a region ofchromosome 12q12-q13.

As used herein, keratin 4 refers to both the gene and the protein unlessclearly indicated otherwise by context. The NCBI Gene ID for humankeratin 4 is 3851 and detailed information can be found at the NCBIwebsite (incorporated herein by reference in the version available onthe filing date of the application to which this application claimspriority). Homo sapiens keratin 4, GenBank Accession No. NM_002272 aminoacid and nucleotide sequences, respectively, are provided in SEQ ID NOs:1 and 2. (The GenBank number is incorporated herein by reference in theversion available on the filing date of the application to which thisapplication claims priority.)

It is understood that the invention includes the use of any fragments ofkeratin 4 polypeptide as long as the fragment allow for the specificidentification of keratin 4 by a detection method of the invention. Forexample, an ELISA antibody must be able to bind to the keratin 4fragment so that detection is possible. Moreover, it is understood thatthere are naturally occurring variants of keratin 4 which may or may notbe associated with a specific disease state, the use of which are alsoincluded in this application. Accordingly, the present inventions alsocontemplates fragments and variants of keratin 4 which may be associatedwith a disease state, e.g., prostate cancer, and/or a particular stageor phase of a disease state, e.g., grades 1-5 of prostate cancer. It isalso understood that the invention encompasses the use of nucleic acidmolecules encoding keratin 4, including for example, keratin 4-encodingDNA, keratin 4 mRNA, and fragments and/or variants thereof. Reference to“keratin 4” may refer to keratin 4 polypeptide or to the keratin 4 gene,unless otherwise indicated.

Keratin 7.

Keratin 7, also known as as CK7, K2C7, K7, SCL, CK-7; cytokeratin 7;cytokeratin-7; keratin, 55K type II cytoskeletal; keratin, simpleepithelial type I, K7; keratin, type II cytoskeletal 7; keratin-7;sarcolectin; type II mesothelial keratin K7; and type-II keratin Kb7, isa member of the keratin gene family. The type II cytokeratins consist ofbasic or neutral proteins which are arranged in pairs of heterotypickeratin chains co-expressed during differentiation of simple andstratified epithelial tissues. This type II cytokeratin is specificallyexpressed in the simple epithelia lining the cavities of the internalorgans and in the gland ducts and blood vessels. The genes encoding thetype II cytokeratins are clustered in a region of chromosome 12q12-q13.Alternative splicing may result in several transcript variants; however,not all variants have been fully described.

As used herein, keratin 7 refers to both the gene and the protein unlessclearly indicated otherwise by context. The NCBI Gene ID for humankeratin 7 is 3855 and detailed information can be found at the NCBIwebsite (incorporated herein by reference in the version available onthe filing date of the application to which this application claimspriority). Homo sapiens keratin 7, GenBank Accession No. NM_005556 aminoacid and nucleotide sequences, respectively, are provided in SEQ ID NOs:3 and 4. (The GenBank number is incorporated herein by reference in theversion available on the filing date of the application to which thisapplication claims priority.)

It is understood that the invention includes the use of any fragments ofkeratin 7 polypeptide as long as the fragment allow for the specificidentification of keratin 7 by a detection method of the invention. Forexample, an ELISA antibody must be able to bind to the keratin 7fragment so that detection is possible. Moreover, it is understood thatthere are naturally occurring variants of keratin 7 which may or may notbe associated with a specific disease state, the use of which are alsoincluded in this application. Accordingly, the present inventions alsocontemplates fragments and variants of keratin 7 which may be associatedwith a disease state, e.g., prostate cancer, and/or a particular stageor phase of a disease state, e.g., grades 1-5 of prostate cancer. It isalso understood that the invention encompasses the use of nucleic acidmolecules encoding keratin 7, including for example, keratin 7-encodingDNA, keratin 7 mRNA, and fragments and/or variants thereof. Reference to“keratin 7” may refer to keratin 7 polypeptide or to the keratin 7 gene,unless otherwise indicated.

Keratin 8.

Keratin 8, also known as K8; KO; CK8; CK-8; CYK8; K2C8; CARD2 is amember of the type II keratin family clustered on the long arm ofchromosome 12. Type I and type II keratins heteropolymerize to formintermediate-sized filaments in the cytoplasm of epithelial cells. Theproduct of this gene typically dimerizes with keratin 18 to form anintermediate filament in simple single-layered epithelial cells. Thisprotein plays a role in maintaining cellular structural integrity andalso functions in signal transduction and cellular differentiation.Mutations in this gene cause cryptogenic cirrhosis. Alternativelyspliced transcript variants have been found for this gene.

As used herein, keratin 8 refers to both the gene and the protein unlessclearly indicated otherwise by context. The NCBI Gene ID for humankeratin 8 is 3856 and detailed information can be found at the NCBIwebsite (incorporated herein by reference in the version available onthe filing date of the application to which this application claimspriority). Homo sapiens keratin 8, variant 1, GenBank Accession No.NM_001256282 amino acid and nucleotide sequences, respectively, areprovided in SEQ ID NOs: 5 and 6; and Homo sapiens keratin 8, variant 3,GenBank Acession No. NM_001256293 amino acid and nucleotide sequences,respectively, are provided in SEQ ID NOs: 7 and 8. (The GenBank numbersare incorporated herein by reference in the version available on thefiling date of the application to which this application claimspriority.)

It is understood that the invention includes the use of any fragments ofkeratin 8 polypeptide as long as the fragment allow for the specificidentification of keratin 8 by a detection method of the invention. Forexample, an ELISA antibody must be able to bind to the keratin 8fragment so that detection is possible. Moreover, it is understood thatthere are naturally occurring variants of keratin 8 which may or may notbe associated with a specific disease state, the use of which are alsoincluded in this application. Accordingly, the present inventions alsocontemplates fragments and variants of keratin 8 which may be associatedwith a disease state, e.g., prostate cancer, and/or a particular stageor phase of a disease state, e.g., grades 1-5 of prostate cancer. It isalso understood that the invention encompasses the use of nucleic acidmolecules encoding keratin 8, including for example, keratin 8-encodingDNA, keratin 8 mRNA, and fragments and/or variants thereof. Reference to“keratin 8” may refer to keratin 8 polypeptide or to the keratin 8 gene,unless otherwise indicated.

Keratin 15.

Keratin 15, also known as as K15; CK15; K1CO, is a member of the keratingene family. The keratins are intermediate filament proteins responsiblefor the structural integrity of epithelial cells and are subdivided intocytokeratins and hair keratins. Most of the type I cytokeratins consistof acidic proteins which are arranged in pairs of heterotypic keratinchains and are clustered in a region on chromosome 17q21.2.

As used herein, keratin 15 refers to both the gene and the proteinunless clearly indicated otherwise by context. The NCBI Gene ID forhuman keratin 15 is 3866 and detailed information can be found at theNCBI website (incorporated herein by reference in the version availableon the filing date of the application to which this application claimspriority). Homo sapiens keratin 15, GenBank Accession No. NM_002275amino acid and nucleotide sequences, respectively, are provided in SEQID NOs: 9 and 10. (The GenBank number is incorporated herein byreference in the version available on the filing date of the applicationto which this application claims priority.)

It is understood that the invention includes the use of any fragments ofkeratin 15 polypeptide as long as the fragment allow for the specificidentification of keratin 15 by a detection method of the invention. Forexample, an ELISA antibody must be able to bind to the keratin 15fragment so that detection is possible. Moreover, it is understood thatthere are naturally occurring variants of keratin 15 which may or maynot be associated with a specific disease state, the use of which arealso included in this application. Accordingly, the present inventionsalso contemplates fragments and variants of keratin 15 which may beassociated with a disease state, e.g., prostate cancer, and/or aparticular stage or phase of a disease state, e.g., grades 1-5 ofprostate cancer. It is also understood that the invention encompassesthe use of nucleic acid molecules encoding keratin 15, including forexample, keratin 15-encoding DNA, keratin 15 mRNA, and fragments and/orvariants thereof. Reference to “keratin 15” may refer to keratin 15polypeptide or to the keratin 15 gene, unless otherwise indicated.

Keratin 18.

Keratin 18, also known as as K18; CYK18, encodes the type I intermediatefilament chain keratin 18. Keratin 18, together with its filamentpartner keratin 8, are perhaps the most commonly found members of theintermediate filament gene family. They are expressed in single layerepithelial tissues of the body. Mutations in this gene have been linkedto cryptogenic cirrhosis. Two transcript variants encoding the sameprotein have been found for this gene.

As used herein, keratin 18 refers to both the gene and the proteinunless clearly indicated otherwise by context. The NCBI Gene ID forhuman keratin 18 is 3875 and detailed information can be found at theNCBI website (incorporated herein by reference in the version availableon the filing date of the application to which this application claimspriority). Homo sapiens keratin 18, variant 1, GenBank Accession No.NM_000224 amino acid and nucleotide sequences, respectively, areprovided in SEQ ID NOs: 11 and 12, and Homo sapiens keratin 18, variant2, GenBank Accession No. 199187 amino acid and nucleotide sequences,respectively, are provided in SEQ ID NOs: 13 and 14. (The GenBanknumbers are incorporated herein by reference in the version available onthe filing date of the application to which this application claimspriority.)

It is understood that the invention includes the use of any fragments ofkeratin 18 polypeptide as long as the fragment allow for the specificidentification of keratin 18 by a detection method of the invention. Forexample, an ELISA antibody must be able to bind to the keratin 18fragment so that detection is possible. Moreover, it is understood thatthere are naturally occurring variants of keratin 18 which may or maynot be associated with a specific disease state, the use of which arealso included in this application. Accordingly, the present inventionsalso contemplates fragments and variants of keratin 18 which may beassociated with a disease state, e.g., prostate cancer, and/or aparticular stage or phase of a disease state, e.g., grades 1-5 ofprostate cancer. It is also understood that the invention encompassesthe use of nucleic acid molecules encoding keratin 18, including forexample, keratin 18-encoding DNA, keratin 18 mRNA, and fragments and/orvariants thereof. Reference to “keratin 18” may refer to keratin 18polypeptide or to the keratin 18 gene, unless otherwise indicated.

Keratin 19.

Keratin 19, also known as K19; CK19; K1CS, is a member of the keratingene family. The keratins are intermediate filament proteins responsiblefor the structural integrity of epithelial cells and are subdivided intocytokeratins and hair keratins. The type I cytokeratins consist ofacidic proteins which are arranged in pairs of heterotypic keratinchains. Unlike its related family members, this smallest known acidiccytokeratin is not paired with a basic cytokeratin in epithelial cells.It is specifically expressed in the periderm, the transientlysuperficial layer that envelopes the developing epidermis. The type Icytokeratins are clustered in a region of chromosome 17q12-q21.

As used herein, keratin 19 refers to both the gene and the proteinunless clearly indicated otherwise by context. The NCBI Gene ID forhuman keratin 19 is 3880 and detailed information can be found at theNCBI website (incorporated herein by reference in the version availableon the filing date of the application to which this application claimspriority). Homo sapiens keratin 19, GenBank Accession No. NM_002276amino acid and nucleotide sequences, respectively, are provided in SEQID NOs: 15 and 16. (The GenBank number is incorporated herein byreference in the version available on the filing date of the applicationto which this application claims priority.)

It is understood that the invention includes the use of any fragments ofkeratin 19 polypeptide as long as the fragment allow for the specificidentification of keratin 19 by a detection method of the invention. Forexample, an ELISA antibody must be able to bind to the keratin 19fragment so that detection is possible. Moreover, it is understood thatthere are naturally occurring variants of keratin 19 which may or maynot be associated with a specific disease state, the use of which arealso included in this application. Accordingly, the present inventionsalso contemplates fragments and variants of keratin 19 which may beassociated with a disease state, e.g., prostate cancer, and/or aparticular stage or phase of a disease state, e.g., grades 1-5 ofprostate cancer. It is also understood that the invention encompassesthe use of nucleic acid molecules encoding keratin 19, including forexample, keratin 19-encoding DNA, keratin 19 mRNA, and fragments and/orvariants thereof. Reference to “keratin 19” may refer to keratin 19polypeptide or to the keratin 8 gene, unless otherwise indicated.

Tubulin-Beta 3.

Tubulin-beta 3, also known as CDCBM; TUBB4; beta-4; CFEOM3A, is a classIII member of the beta tubulin protein family. Beta tubulins are one oftwo core protein families (alpha and beta tubulins) that heterodimerizeand assemble to form microtubules. This protein is primarily expressedin neurons and may be involved in neurogenesis and axon guidance andmaintenance. Mutations in this gene are the cause of congenital fibrosisof the extraocular muscles type 3. Alternate splicing results inmultiple transcript variants. A pseudogene of this gene is found onchromosome 6.

As used herein, Tubulin-beta 3 refers to both the gene and the proteinunless clearly indicated otherwise by context. The NCBI Gene ID forhuman Tubulin-beta 3 is 10381 and detailed information can be found atthe NCBI website (incorporated herein by reference in the versionavailable on the filing date of the application to which thisapplication claims priority). Homo sapiens Tubulin-beta 3, variant 2,GenBank Accession No. NM_001197181 amino acid and nucleotide sequences,respectively, are provided in SEQ ID NOs: 17 and 18. Homo sapiensTubulin-beta 3, variant 1, GenBank Accession No. NM_006086 amino acidand nucleotide sequences, respectively, are provided in SEQ ID NOs: 19and 20. (The GenBank numbers are incorporated herein by reference in theversions available on the filing date of the application to which thisapplication claims priority.)

It is understood that the invention includes the use of any fragments ofTubulin-beta 3 polypeptide as long as the fragment allow for thespecific identification of Tubulin-beta 3 by a detection method of theinvention. For example, an ELISA antibody must be able to bind to theTubulin-beta 3 fragment so that detection is possible. Moreover, it isunderstood that there are naturally occurring variants of Tubulin-beta 3which may or may not be associated with a specific disease state, theuse of which are also included in this application. Accordingly, thepresent inventions also contemplates fragments and variants ofTubulin-beta 3 which may be associated with a disease state, e.g.,prostate cancer, and/or a particular stage or phase of a disease state,e.g., grades 1-5 of prostate cancer. It is also understood that theinvention encompasses the use of nucleic acid molecules encodingTubulin-beta 3, including for example, Tubulin-beta 3-encoding DNA,Tubulin-beta 3 mRNA, and fragments and/or variants thereof. Reference to“Tubulin-beta 3” may refer to Tubulin-beta 3 polypeptide or to theTubulin-beta 3 gene, unless otherwise indicated.

Filamin B.

Filamin B is also known as filamin-3, beta-filamin, ABP-280 homolog,filamin homolog 1, thyroid autoantigen, actin binding protein 278,actin-binding-like protein, Larsen syndrome 1 (autosomal dominant), AOI;FH1; SCT; TAP; LRS1; TABP; FLN-B; FLN1L; ABP-278; and ABP-280. The geneencodes a member of the filamin family. The encoded protein interactswith glycoprotein Ib alpha as part of the process to repair vascularinjuries. The platelet glycoprotein Ib complex includes glycoprotein Ibalpha, and it binds the actin cytoskeleton. Mutations in this gene havebeen found in several conditions: atelosteogenesis type 1 and type 3;boomerang dysplasia; autosomal dominant Larsen syndrome; andspondylocarpotarsal synostosis syndrome. Multiple alternatively splicedtranscript variants that encode different protein isoforms have beendescribed for this gene.

As used herein, filamin B refers to both the gene and the protein unlessclearly indicated otherwise by context. The NCBI gene ID for filamin Bis 2317 and detailed information can be found at the NCBI website(incorporated herein by reference in the version available on the filingdate of the application to which this application claims priority).

Homo sapiens filamin B, beta (FLNB), RefSeqGene on chromosome 3, locusNG_012801 is shown in SEQ ID NO: 21. Homo sapiens filamin B, beta(FLNB), transcript variant 1, GenBank Accession No. NM_001164317.1 aminoacid and nucleotide sequences, respectively, are provided in SEQ ID NOs:22 and 23. Homo sapiens filamin B, beta (FLNB), transcript variant 3,GenBank Accession No. NM_001164318.1 amino acid and nucleotidesequences, respectively, are provided in SEQ ID NOs: 24 and 25. Homosapiens filamin B, beta (FLNB), transcript variant 4, GenBank AccessionNo. NM_001164319.1 amino acid and nucleotide sequences, respectively,are provided in SEQ ID NOs: 26 and 27. Homo sapiens filamin B, beta(FLNB), transcript variant 2, GenBank Accession No. NM_001457.3 aminoacid and nucleotide sequences, respectively, are provided in SEQ ID NOs:28 and 29. (Each GenBank number is incorporated herein by reference inthe version available on the filing date of the application to whichthis application claims priority.)

It is understood that the invention includes the use of any combinationof one or more of the filamin B sequences provided in the sequencelisting or any fragments thereof as long as the fragment can allow forthe specific identification of filamin B. Methods of the invention andreagents can be used to detect single isoforms of filamin B,combinations of filamin B isoforms, or all of the filamin B isoformssimultaneously. Unless specified, filamin B can be considered to referto one or more isoforms of filamin B, including total filamin B.Moreover, it is understood that there are naturally occurring variantsof filamin B, which may or may not be associated with a specific diseasestate, the use of which are also included in the instant application.

In addition, it is understood that the invention includes the use of anyfragments of filamin B polypeptide as long as the fragment allow for thespecific identification of filamin B by a detection method of theinvention. For example, an ELISA antibody must be able to bind to thefilamin B fragment so that detection is possible. Moreover, it isunderstood that there are naturally occurring variants of filamin Bwhich may or may not be associated with a specific disease state, theuse of which are also included in this application. Accordingly, thepresent inventions also contemplates fragments and variants of filamin Bwhich may be associated with a disease state, e.g., prostate cancer,and/or a particular stage or phase of a disease state, e.g., grades 1-5of prostate cancer. It is also understood that the invention encompassesthe use of nucleic acid molecules encoding filamin B, including forexample, filamin B-encoding DNA, filamin B mRNA, and fragments and/orvariants thereof. Reference to “filamin B” may refer to filamin Bpolypeptide or to the filamin B gene, unless otherwise indicated.

Lymphocyte Antigen 9.

Lymphocyte antigen 9 (LY9) is also known as RP11-312J18.1, CD229,SLAMF3, hly9, mLY9, T-lymphocyte surface antigen Ly-9; and cell surfacemolecule Ly-9. LY9 belongs to the SLAM family of immunomodulatoryreceptors (see SLAMF1; MIM 603492) and interacts with the adaptormolecule SAP (SH2D1A; MIM 300490) (Graham et al., 2006).

As used herein, LY9 refers to both the gene and the protein unlessclearly indicated otherwise by context. The NCBI gene ID for LY9 is 4063and detailed information can be found at the NCBI website (incorporatedherein by reference in the version available on the filing date of theapplication to which this application claims priority). Homo sapienslymphocyte antigen 9 (LY9), transcript variant 2, GenBank Accession No.NM_001033667 amino acid and nucleotide sequences, respectively, areprovided in SEQ ID NOs: 30 and 31. Homo sapiens lymphocyte antigen 9(LY9), transcript variant 3, GenBank Accession No. NM_001261456 aminoacid and nucleotide sequences, respectively, are provided in SEQ ID NOs:32 and 33. Homo sapiens lymphocyte antigen 9 (LY9), transcript variant4, GenBank Accession No. NM_001261457 amino acid and nucleotidesequences, respectively, are provided in SEQ ID NOs: 34 and 35. Homosapiens lymphocyte antigen 9 (LY9), transcript variant 1, GenBankAccession No. NM_002348 is shown amino acid and nucleotide sequences,respectively, are provided in SEQ ID NOs: 36 and 37. (Each GenBanknumber is incorporated herein by reference in the version available onthe filing date of the application to which this application claimspriority.)

It is understood that the invention includes the use of any combinationof one or more of the LY9 sequences provided in the sequence listing orany fragments thereof as long as the fragment can allow for the specificidentification of LY9. Methods of the invention and reagents can be usedto detect single isoforms of LY9, combinations of LY9 isoforms, or allof the LY9 isoforms simultaneously. Unless specified, LY9 can beconsidered to refer to one or more isoforms of LY9, including total LY9.Moreover, it is understood that there are naturally occurring variantsof LY9, which may or may not be associated with a specific diseasestate, the use of which are also included in the instant application.

In addition, it is understood that the invention includes the use of anyfragments of LY9 polypeptide as long as the fragment allow for thespecific identification of LY9 by a detection method of the invention.For example, an ELISA antibody must be able to bind to the LY9 fragmentso that detection is possible. Moreover, it is understood that there arenaturally occurring variants of LY9 which may or may not be associatedwith a specific disease state, the use of which are also included inthis application. Accordingly, the present inventions also contemplatesfragments and variants of LY9 which may be associated with a diseasestate, e.g., prostate cancer, and/or a particular stage or phase of adisease state, e.g., grades 1-5 of prostate cancer. It is alsounderstood that the invention encompasses the use of nucleic acidmolecules encoding LY9, including for example, LY9-encoding DNA, LY9mRNA, and fragments and/or variants thereof. Reference to “LY9” mayrefer to LY9 polypeptide or to the LY9 gene, unless otherwise indicated.

Prostate Specific Antigen.

Prostate-specific antigen (PSA) is also known as kallikrein-3, seminin,P-30 antigen, semenogelase, gamma-seminoprotein, APS, hK3, and KLK2A1.Kallikreins are a subgroup of serine proteases having diversephysiological functions. Growing evidence suggests that many kallikreinsare implicated in carcinogenesis and some have potential as novel cancerand other disease biomarkers. This gene is one of the fifteen kallikreinsubfamily members located in a cluster on chromosome 19. Its proteinproduct is a protease present in seminal plasma. It is thought tofunction normally in the liquefaction of seminal coagulum, presumably byhydrolysis of the high molecular mass seminal vesicle protein. Serumlevel of this protein, called PSA in the clinical setting, is useful inthe diagnosis and monitoring of prostatic carcinoma. Alternate splicingof this gene generates several transcript variants encoding differentisoforms.

As used herein, PSA refers to both the gene and the protein, in bothprocessed and unprocessed forms, unless clearly indicated otherwise bycontext. The NCBI gene ID for PSA is 354 and detailed information can befound at the NCBI website (incorporated herein by reference in theversion available on the filing date of the application to which thisapplication claims priority).

Homo sapiens PSA is located on chromosome 19 at 19q13.41Sequence:NC_000019.9 (51358171 . . . 51364020). Four splice variants of human PSAare known. Prostate-specific antigen isoform 3 preproprotein,NM_001030047.1, is provided as SEQ ID NOs: 38 and 39. Prostate-specificantigen isoform 4 preproprotein, NM_001030048.1, is provided as SEQ IDNOs: 40 and 41. Prostate-specific antigen isoform 6 preproprotein,NM_001030050.1, is provided as SEQ ID NOs: 42 and 43. Prostate-specificantigen isoform 1 preproprotein, NM_001648.2, is provided in SEQ ID NOs:44 and 45. (Each GenBank number is incorporated herein by reference inthe version available on the filing date of the application to whichthis application claims priority).

It is understood that the invention includes the use of any combinationof one or more of the PSA sequences provided in the sequence listing orany fragments thereof as long as the fragment can allow for the specificidentification of PSA. Methods of the invention and reagents can be usedto detect single isoforms of PSA, combinations of PSA isoforms, or allof the PSA isoforms simultaneously. Unless specified, PSA can beconsidered to refer to one or more isoforms of PSA, including total PSA.Moreover, it is understood that there are naturally occurring variantsof PSA, which may or may not be associated with a specific diseasestate, the use of which are also included in the instant application.

In addition, it is understood that the invention includes the use of anyfragments of PSA polypeptide as long as the fragment allow for thespecific identification of PSA by a detection method of the invention.For example, an ELISA antibody must be able to bind to the PSA fragmentso that detection is possible. Moreover, it is understood that there arenaturally occurring variants of PSA which may or may not be associatedwith a specific disease state, the use of which are also included inthis application. Accordingly, the present inventions also contemplatesfragments and variants of PSA which may be associated with a diseasestate, e.g., prostate cancer, and/or a particular stage or phase of adisease state, e.g., grades 1-5 of prostate cancer. It is alsounderstood that the invention encompasses the use of nucleic acidmolecules encoding PSA, including for example, PSA-encoding DNA, PSAmRNA, and fragments and/or variants thereof. Reference to “PSA” mayrefer to PSA polypeptide or to the PSA gene, unless otherwise indicated.

Age.

The age of a subject can be used as a continuous predictive variable forthe presence of prostate cancer. For example, increased age isassociated with increased risk of prostate cancer. Conversely, decreasedage is associated with decreased risk of prostate cancer. Similarly, agecan be used as a continuous predictive variable for the stage, orcategory, of the prostate cancer. For example, age can be used as acontinuous predictive variable for the Gleason score of the prostatecancer.

The biomarkers of the invention, including in particular filamin A aloneor in combination with any one or more of PSA, keratin 4, keratin 7,keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, filamin B(FLNB), and lymphocyte antigen 9 (LY9), may be detected as a polypeptideor a detectable fragment thereof. Alternatively, the biomarkers of theinvention, including in particular filamin A alone or in combinationwith any one or more of PSA, keratin 4, keratin 7, keratin 8, keratin15, keratin 18, keratin 19, tubulin-beta 3, filamin B (FLNB), andlymphocyte antigen 9 (LY9), may be detected as nucleic acid molecules,such as DNA, RNA, mRNA, microRNA, and the like. In addition,combinations of biomarkers, including filamin A alone or in combinationwith any one or more of PSA, keratin 4, keratin 7, keratin 8, keratin15, keratin 18, keratin 19, tubulin-beta 3, filamin B (FLNB), andlymphocyte antigen 9 (LY9), may be detected as any combination ofpolypeptides and nucleic acid molecules. In certain embodiments, all ofthe biomarkers are in the form of polypeptides. In certain otherembodiments, all of the biomarkers are in the form of polynucleotides.In certain other embodiments, at least filamin A is in the form of apolypeptide, whereas any other markers tested can be a polypeptide ornucleic acid molecule. In still other embodiments, at least filamin A isin the form of a nucleic acid molecule, whereas any other markers testedcan be a polypeptide or nucleic acid molecule.

In other embodiments, the biomarkers of the invention, including inparticular filamin A alone or in combination with any one or more ofPSA, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin19, tubulin-beta 3, filamin B (FLNB), lymphocyte antigen 9 (LY9),prostate-specific membrane antigen (PSM), prostate stem cell antigen(PSCA), TMPRSS2, PDEF, prostate-specific gene-1 (HPG-1), and non-codingRNAs (ncRNAs), including PCA3, PCGEM1, and the gene cluster P704P,P712P, and P775P, may be detected as a polypeptide or a detectablefragment thereof. Alternatively, the biomarkers of the invention,including in particular filamin A alone or in combination with any oneor more of PSA, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, tubulin-beta 3, filamin B (FLNB), lymphocyte antigen 9(LY9), prostate-specific membrane antigen (PSM), prostate stem cellantigen (PSCA), TMPRSS2, PDEF, prostate-specific gene-1 (HPG-1), andnon-coding RNAs (ncRNAs), including PCA3, PCGEM1, and the gene clusterP704P, P712P, and P775P, may be detected as nucleic acid molecules, suchas DNA, RNA, mRNA, microRNA, and the like. In addition, combinations ofbiomarkers, including filamin A alone or in combination with any one ormore of PSA, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, tubulin-beta 3, filamin B (FLNB), lymphocyte antigen 9(LY9), prostate-specific membrane antigen (PSM), prostate stem cellantigen (PSCA), TMPRSS2, PDEF, prostate-specific gene-1 (HPG-1), andnon-coding RNAs (ncRNAs), including PCA3, PCGEM1, and the gene clusterP704P, P712P, and P775P, may be detected as any combination ofpolypeptides and nucleic acid molecules. In certain embodiments, all ofthe biomarkers are in the form of polypeptides. In certain otherembodiments, all of the biomarkers are in the form of polynucleotides.In certain other embodiments, at least filamin A is in the form of apolypeptide, whereas any other markers tested can be a polypeptide ornucleic acid molecule. In still other embodiments, at least filamin A isin the form of a nucleic acid molecule, whereas any other markers testedcan be a polypeptide or nucleic acid molecule.

The specific marker identified herein as prostate-specific membraneantigen (PSM) is further described in Sokoll et al., 1997,Prostate-specific antigen—Its discovery and biochemical characteristics,Urol. Clin. North Am., 24:253-259, which is incorporated herein byreference.

The specific marker identified herein as prostate stem cell antigen(PSCA) is further described in Fair et al., 1997, Prostate-specificmembrane antigen, Prostate, 32:140-148, which is incorporated herein byreference.

The specific marker identified herein as TMPRSS2 is further described inLin et al., 1999, Prostate-localized and androgen-regulated expressionof the membrane-bound serine protease TMPRSS2, Cancer Res.,59:4180-4184, which is incorporated herein by reference.

The specific marker identified herein as PDEF is further described inOettgen et al., PDEF, a novel prostate epithelium-specific ETStranscription factor interacts with the androgen receptor and activatesprostate-specific antigen gene expression, J. Biol. Chem., 275:1216-1225, which is incorporated herein by reference.

The specific marker identified herein as prostate-specific gene-1(HPG-1) is further described in Herness, A novel human prostate-specificgene-1 (HPG-1): molecular cloning, sequencing, and its potentialinvolvement in prostate carcinogenesis, 2003, Cancer Res. 63:329-336,which is incorporated herein by reference.

The non-coding RNA's (ncRNA's) identified as PCA3 is further describedin Bussemakers et al., 1999, DD3: a new prostate-specific gene, highlyoverexpressed in prostate cancer, Cancer Res. 59:5975-5979, which isincorporated herein by reference.

The non-coding RNA identified as PCGEM1 is further described inSrikantan et al., 2000. PCGEM1, a prostate-specific gene, isoverexpressed in prostate cancer. Proc. Natl. Acad. Sci. USA97:12216-12221, which is incorporated herein by reference.

The gene cluster P704P, P712P, and P775P is further described in Stolket al., 2004. P704P, P712P, and P775P: A genomic cluster ofprostate-specific genes. Prostate 60:214-226), which is incorporatedherein by reference.

The present invention also contemplates the use of particularcombinations of biomarkers.

In one embodiment, the invention contemplates marker sets with at leasttwo (2) members, which may include, but are not limited to the followingsets: filamin A together with PSA; filamin A together with PSA; filaminA together with LY9; filamin A together with keratin 4; filamin Atogether with kertain 7; filamin A together with keratin 8; filamin Atogether with keratin 15; filamin A together with keratin 18; filamin Atogether with keratin 19; filamin A together with tubulin-beta 3;filamin A together with prostate-specific membrane antigen (PSM);filamin A together with prostate stem cell antigen (PSCA); filamin Atogether with TMPRSS2; filamin A together with PDEF; filamin A togetherwith prostate-specific gene-1 (HPG-1); filamin A together with PCA3;filamin A together with PCGEM1; and filamin A together with gene clusterP704P, P712P, and P775P; and filamin A together with patient age. Anymarker set can additionally be used in combination with PSA.

In another embodiment, the invention contemplates marker sets with atleast three (3) members, wherein one member is filamin A and theadditional two members are selected from the following sets of twomarkers: filamin B, LY9; filamin B, keratin 4; filamin B, keratin 7;filamin B, keratin 8; filamin B, keratin 15; filamin B, keratin 18;filamin B, keratin 19; filamin B, tubulin-beta 3; filamin B, PSM;filamin B, PSCA; filamin B, TMPRSS2; filamin B, PDEF; filamin B, HPG-1;filamin B, PCA3; filamin B, PCGEM1; filamin B, P704P/P712P/P775P; LY9,keratin 4; LY9, keratin 7; LY9, keratin 8; LY9, keratin 15; LY9, keratin18; LY9, keratin 19; LY9, tubulin-beta 3; LY9, PSM; LY9, PSCA; LY9,TMPRSS2; LY9, PDEF; LY9, HPG-1; LY9, PCA3; LY9, PCGEM1; LY9,P704P/P712P/P775P; keratin 4, keratin 7; keratin 4, keratin 8; keratin4, keratin 15; keratin 4, keratin 18; keratin 4, keratin 19; keratin 4,tubulin-beta 3; keratin 4, PSM; keratin 4, PSCA; keratin 4, TMPRSS2;keratin 4, PDEF; keratin 4, HPG-1; keratin 4, PCA3; keratin 4, PCGEM1;keratin 4, P704P/P712P/P775P; keratin 7, keratin 8; keratin 7, keratin15; keratin 7, keratin 18; keratin 7, keratin 19; keratin 7,tubulin-beta 3; keratin 7, PSM; keratin 7, PSCA; keratin 7, TMPRSS2;keratin 7, PDEF; keratin 7, HPG-1; keratin 7, PCA3; keratin 7, PCGEM1;keratin 7, P704P/P712P/P775P; keratin 8, keratin 15; keratin 8, keratin18; keratin 8, keratin 19; keratin 8, tubulin-beta 3; keratin 8, PSM;keratin 8, PSCA; keratin 8, TMPRSS2; keratin 8, PDEF; keratin 8, HPG-1;keratin 8, PCA3; keratin 8, PCGEM1; keratin 8, P704P/P712P/P775P;keratin 15, keratin 18; keratin 15, keratin 19; keratin 15, tubulin-beta3; keratin 15, PSM; keratin 15, PSCA; keratin 15, TMPRSS2; keratin 15,PDEF; keratin 15, HPG-1; keratin 15, PCA3; keratin 15, PCGEM1; keratin15, P704P/P712P/P775P; keratin 18, tubulin-beta 3; keratin 18, keratin19; and keratin 19, tubulin-beta 3; keratin 18, PSM; keratin 18, PSCA;keratin 18, TMPRSS2; keratin 18, PDEF; keratin 18, HPG-1; keratin 18,PCA3; keratin 18, PCGEM1; keratin 18, P704P/P712P/P775P. Any marker setcan be used in combination with patient age. Any marker set canadditionally be used in combination with PSA.

In another embodiment, the invention contemplates marker sets with atleast four (4) members, wherein one member is filamin A and theadditional three members may include, but are not limited to thefollowing sets: filamin B, LY9, keratin 4; filamin B, LY9, keratin 7;filamin B, LY9, keratin 8; filamin B, LY9, keratin 15; filamin B, LY9,keratin 18; filamin B, LY9, keratin 19; filamin B, LY9, tubulin-beta 3;filamin B, keratin 4, keratin 7; filamin B, keratin 4, keratin 8;filamin B, keratin 4, keratin 15; filamin B, keratin 4, keratin 18;filamin B, keratin 4, keratin 19; filamin B, keratin 4, tubulin-beta 3;filamin B, keratin 7, keratin 8; filamin B, keratin 7, keratin 15;filamin B, keratin 7, keratin 18; filamin B, keratin 7, keratin 19;filamin B, keratin 7, tubulin-beta 3; filamin B, keratin 8, keratin 15;filamin B, keratin 8, keratin 18; filamin B, keratin 8, keratin 19;filamin B, keratin 8, tubulin-beta 3; filamin B, keratin 15, keratin 18;filamin B, keratin 15, keratin 19; filamin B, keratin 15, tubulin-beta3; filamin B, keratin 18, keratin 19; filamin B, keratin 18,tubulin-beta 3; filamin B, keratin 19, tubulin-beta 3; LY9, keratin 4,keratin 7; LY9, keratin 4, keratin 8; LY9, keratin 4, keratin 15; LY9,keratin 4, keratin 18; LY9, keratin 4, keratin 19; LY9, keratin 4,tubulin-beta 3; LY9, keratin 7, keratin 8; LY9, keratin 7, keratin 15;LY9, keratin 7, keratin 18; LY9, keratin 7, keratin 19; LY9, keratin 7,tubulin-beta 3; LY9, keratin 8, keratin 15; LY9, keratin 8, keratin 18;LY9, keratin 8, keratin 19; LY9, keratin 8, tubulin-beta 3; LY9, keratin15, keratin 18; LY9, keratin 15, keratin 19; LY9, keratin 15,tubulin-beta 3; LY9, keratin 18, keratin 19; LY9, keratin 18,tubulin-beta 3; LY9, keratin 19, tubulin-beta 3; keratin 4, keratin 7,keratin 8; keratin 4, keratin 7, keratin 15; keratin 4, keratin 7,keratin 18; keratin 4, keratin 7, keratin 19; keratin 4, keratin 7,tubulin-beta 3; keratin 4, keratin 8, keratin 15; keratin 4, keratin 8,keratin 18; keratin 4, keratin 8, keratin 19; keratin 4, keratin 8,tubulin-beta 3; keratin 4, keratin 15, keratin 18; keratin 4, keratin15, keratin 19; keratin 4, keratin 15, tubulin-beta 3; keratin 4,keratin 18, keratin 19; keratin 4, keratin 19, tubulin-beta 3; keratin7, keratin 8, keratin 15; keratin 7, keratin 8, keratin 18; keratin 7,keratin 8, keratin 19; keratin 7, keratin 8, tubulin-beta 3; keratin 7,keratin 8, tubulin-beta 3; keratin 7, keratin 15, keratin 18; keratin 7,keratin 15, keratin 19; keratin 7, keratin 15, tubulin-beta 3; keratin7, keratin 18, keratin 19; keratin 7, keratin 18, tubulin-beta 3;keratin 15, keratin 18, keratin 19; keratin 15, keratin 18, tubulin-beta3; and keratin 18, keratin 19, tubulin-beta 3. Any marker set can beused in combination with patient age. Any marker set can be used incombination with PSA. In addition, any of the above sets may be modifiedto replace one or more markers in the marker set with one or more of thefollowing additional markers: prostate-specific membrane antigen (PSM),prostate stem cell antigen (PSCA), TMPRSS2, PDEF, prostate-specificgene-1 (HPG-1), PCA3, PCGEM1, and the gene cluster P704P, P712P, andP775P.

In another embodiment, the invention contemplates marker sets with atleast five (5) members, wherein one member is filamin A and theadditional four members may include, but are not limited to thefollowing sets: filamin B, LY9, keratin 4, keratin 7; filamin B, LY9,keratin 4, keratin 8; filamin B, LY9, keratin 4, keratin 15; filamin B,LY9, keratin 4, keratin 18; filamin B, LY9, keratin 4, keratin 19;filamin B, LY9, keratin 4, tubulin-beta 3; filamin B, keratin 4, keratin7, keratin 8; filamin B, keratin 4, keratin 7, keratin 15; filamin B,keratin 4, keratin 7, keratin 18; filamin B, keratin 4, keratin 7,tubulin-beta 3; filamin B, keratin 4, keratin 7, tubulin-beta 3; filaminB, keratin 7, keratin 8, keratin 15; filamin B, keratin 7, keratin 8,keratin 18; filamin B, keratin 7, keratin 8, keratin 19; filamin B,keratin 7, keratin 8, tubulin-beta 3; filamin B, keratin 8, keratin 15,keratin 18; filamin B, keratin 8, keratin 15, keratin 19; filamin B,keratin 8, keratin 15, tubulin-beta 3; filamin B, keratin 15, keratin18, keratin 19; filamin B, keratin 15, keratin 18, tubulin-beta 3;filamin B, keratin 18, keratin 19, and tubulin-beta 3; LY9, keratin 4,keratin 7, keratin 8; LY9, keratin 4, keratin 7, keratin 15; LY9,keratin 4, keratin 7, keratin 18; LY9, keratin 4, keratin 7, keratin 19;LY9, keratin 4, keratin 7, tubulin-beta 3; LY9, keratin 7, keratin 8,keratin 15; LY9, keratin 7, keratin 8, keratin 18; LY9, keratin 7,keratin 8, keratin 19; LY9, keratin 7, keratin 8, tubulin-beta 3; LY9,keratin 8, keratin 15, keratin 18; LY9, keratin 8, keratin 15, keratin19; LY9, keratin 8, keratin 15, tubulin-beta 3; LY9, keratin 15, keratin18, keratin 19; LY9, keratin 15, keratin 18, tubulin-beta 3; LY9,keratin 18, keratin 19, and tubulin-beta 3; keratin 4, keratin 7,keratin 8, keratin 15; keratin 4, keratin 7, keratin 8, keratin 18;keratin 4, keratin 7, keratin 8, keratin 19; keratin 4, keratin 7,keratin 8, tubulin-beta 3; keratin 4, keratin 8, keratin 15, keratin 18;keratin 4, keratin 8, keratin 15, keratin 19; keratin 4, keratin 8,keratin 15, tubulin-beta 3; keratin 4, keratin 15, keratin 18, keratin19; keratin 4, keratin 15, keratin 18, tubulin-beta 3; keratin 4,keratin 18, keratin 19, tubulin-beta 3; keratin 8, keratin 15, keratin18, keratin 19; keratin 8, keratin 15, keratin 18, tubulin-beta 3; andkeratin 15, keratin 18, keratin 19, tubulin-beta 3. Any marker set canbe used in combination with PSA. In addition, any of the above sets maybe modified to replace one or more markers in the marker set with one ormore of the following additional markers: prostate-specific membraneantigen (PSM), prostate stem cell antigen (PSCA), TMPRSS2, PDEF,prostate-specific gene-1 (HPG-1), PCA3, PCGEM1, and the gene clusterP704P, P712P, and P775P. Any marker set can be used in combination withpatient age.

In another embodiment, the invention contemplates marker sets with atleast six (6) members, wherein one member is filamin A and theadditional five members may include, but are not limited to thefollowing sets: keratin 8, keratin 15, keratin 18, keratin 19tubulin-beta 3; keratin 7, keratin 15, keratin 18, keratin 19tubulin-beta 3; keratin 7, keratin 8, keratin 18, keratin 19tubulin-beta 3; keratin 7, keratin 8, keratin 15, keratin 19tubulin-beta 3; keratin 7, keratin 8, keratin 15, keratin 18tubulin-beta 3; keratin 7, keratin 8, keratin 15, keratin 18, keratin19; keratin 4, keratin 15, keratin 18, keratin 19 tubulin-beta 3;keratin 4, keratin 8, keratin 18, keratin 19 tubulin-beta 3; keratin 4,keratin 8, keratin 15, keratin 19 tubulin-beta 3; keratin 4, keratin 8,keratin 15, keratin 18 tubulin-beta 3; keratin 4, keratin 8, keratin 15,keratin 18, keratin 19; LY9, keratin 15, keratin 18, keratin 19,tubulin-beta 3; LY9, keratin 8, keratin 18, keratin 19 tubulin-beta 3;LY9, keratin 8, keratin 15, keratin 19 tubulin-beta 3; LY9, keratin 8,keratin 15, keratin 18, and tubulin-beta 3; LY9, keratin 8, keratin 15,keratin 18, keratin 19; filamin B, keratin 15, keratin 18, keratin 19tubulin-beta 3; filamin B, keratin 8, keratin 18, keratin 19tubulin-beta 3; filamin B, keratin 8, keratin 15, keratin 19tubulin-beta 3; filamin B, keratin 8, keratin 15, keratin 18, andtubulin-beta 3; filamin B, keratin 8, keratin 15, keratin 18, keratin19; filamen B, LY9, keratin 18, keratin 19 tubulin-beta 3; filamen B,LY9, keratin 15, keratin 19 tubulin-beta 3; filamen B, LY9, keratin 15,keratin 18, tubulin-beta 3; filamen B, LY9, keratin 15, keratin 18,keratin 19; filamen B, keratin 4, keratin 18, keratin 19 tubulin-beta 3;filamen B, keratin 4, keratin 15, keratin 19 tubulin-beta 3; filamen B,keratin 4, keratin 15, keratin 18, tubulin-beta 3; filamen B, keratin 4,keratin 15, keratin 18, keratin 19; filamen B keratin 7, keratin 18,keratin 19 tubulin-beta 3; filamen B keratin 7, keratin 15, keratin 19,tubulin-beta 3; filamen B keratin 7, keratin 15, keratin 18,tubulin-beta 3; filamen B keratin 7, keratin 15, keratin 18, keratin 19;filamen B, keratin 8, keratin 18, keratin 19 tubulin-beta 3; filamen B,keratin 8, keratin 15, keratin 19 tubulin-beta 3; filamen B, keratin 8,keratin 15, keratin 18 tubulin-beta 3; filamen B, keratin 8, keratin 15,keratin 18, keratin 19; LY9, keratin 4, keratin 18, keratin 19 andtubulin-beta 3; LY9, keratin 4, keratin 15, keratin 19 tubulin-beta 3;LY9, keratin 4, keratin 15, keratin 18, tubulin-beta 3; LY9, keratin 4,keratin 15, keratin 18, keratin 19; LY9, keratin 7, keratin 18, keratin19 tubulin-beta 3; LY9, keratin 7, keratin 15, keratin 19 tubulin-beta3; LY9, keratin 7, keratin 15, keratin 18, and tubulin-beta 3; LY9,keratin 7, keratin 15, keratin 18, keratin 19; LY9, keratin 8, keratin18, keratin 19 tubulin-beta 3; LY9, keratin 8, keratin 15, keratin 19tubulin-beta 3; LY9, keratin 8, keratin 15, keratin 18, and tubulin-beta3; LY9, keratin 8, keratin 15, keratin 18, keratin 19; keratin 4,keratin 7, keratin 18, keratin 19 tubulin-beta 3; keratin 4, keratin 7,keratin 15, keratin 19 tubulin-beta 3; keratin 4, keratin 7, keratin 15,keratin 18, and tubulin-beta 3; keratin 4, keratin 7, keratin 15,keratin 18, keratin 19; keratin 4, keratin 8, keratin 18, keratin 19tubulin-beta 3; keratin 4, keratin 8, keratin 15, keratin 19tubulin-beta 3; keratin 4, keratin 8, keratin 15, keratin 18, andtubulin-beta 3; keratin 4, keratin 8, keratin 15, keratin 18, keratin19; keratin 7, keratin 8, keratin 18, keratin 19 tubulin-beta 3; keratin7, keratin 8, keratin 15, keratin 19 tubulin-beta 3; keratin 7, keratin8, keratin 15, keratin 18, and tubulin-beta 3; keratin 7, keratin 8,keratin 15, keratin 18, keratin 19; filamen B, LY9, keratin 4, keratin19, tubulin-beta 3; filamen B, LY9, keratin 4, keratin 18, ubulin-beta3; filamen B, LY9, keratin 4, keratin 18, keratin 19; filamen B, LY9,keratin 7, keratin 19, tubulin-beta 3; filamen B, LY9, keratin 7,keratin 18, tubulin-beta 3; filamen B, LY9, keratin 7, keratin 18,keratin 19; filamen B, LY9, keratin 8, keratin 19, tubulin-beta 3;filamen B, LY9, keratin 8, keratin 18, tubulin-beta 3; filamen B, LY9,keratin 8, keratin 18, keratin 19; filamen B, LY9, keratin 15, keratin19, tubulin-beta 3; filamen B, LY9, keratin 15, keratin 18, tubulin-beta3; filamen B, LY9, keratin 15, keratin 18, keratin 19; filamen B,keratin 4, keratin 7, keratin 19, tubulin-beta 3; filamen B, keratin 4,keratin 7, keratin 18, tubulin-beta 3; filamen B, keratin 4, keratin 7,keratin 18, keratin 19; filamen B, keratin 4, keratin 8, keratin 19,tubulin-beta 3; filamen B, keratin 4, keratin 8, keratin 18,tubulin-beta 3; filamen B, keratin 4, keratin 8, keratin 18, keratin 19;filamen B, keratin 4, keratin 15, keratin 19, tubulin-beta 3; filamen B,keratin 4, keratin 15, keratin 18, tubulin-beta 3; filamen B, keratin 4,keratin 15, keratin 18, keratin 19; filamen B, keratin 7, keratin 8,keratin 19, tubulin-beta 3; filamen B, keratin 7, keratin 8, keratin 18,tubulin-beta 3; filamen B, keratin 7, keratin 8, keratin 18, keratin 19;filamen B, keratin 8, keratin 15, keratin 19, tubulin-beta 3; filamen B,keratin 8, keratin 15, keratin 18, tubulin-beta 3; filamen B, keratin 8,keratin 15, keratin 18, keratin 19; LY9, keratin 4, keratin 7, keratin19, tubulin-beta 3; LY9, keratin 4, keratin 7, keratin 18, tubulin-beta3; LY9, keratin 4, keratin 7, keratin 18, keratin 19; LY9, keratin 4,keratin 8, keratin 19, tubulin-beta 3; LY9, keratin 4, keratin 8,keratin 18, tubulin-beta 3; LY9, keratin 4, keratin 8, keratin 18,keratin 19; LY9, keratin 4, keratin 15, keratin 19, tubulin-beta 3; LY9,keratin 4, keratin 15, keratin 18, tubulin-beta 3; LY9, keratin 4,keratin 15, keratin 18, keratin 19; LY9, keratin 7, keratin 8, keratin19, tubulin-beta 3; LY9, keratin 7, keratin 8, keratin 18, tubulin-beta3; LY9, keratin 7, keratin 8, keratin 18, keratin 19; LY9, keratin 7,keratin 15, keratin 19, tubulin-beta 3; LY9, keratin 7, keratin 15,keratin 18, tubulin-beta 3; LY9, keratin 7, keratin 15, keratin 18,keratin 19; LY9, keratin 8, keratin 15, keratin 19, tubulin-beta 3; LY9,keratin 8, keratin 15, keratin 18, tubulin-beta 3; LY9, keratin 8,keratin 15, keratin 18, keratin 19; keratin 4, keratin 7, keratin 8,keratin 19, tubulin-beta 3; keratin 4, keratin 7, keratin 8, keratin 18,tubulin-beta 3; keratin 4, keratin 7, keratin 8, keratin 18, keratin 19;keratin 4, keratin 7, keratin 15, keratin 19, tubulin-beta 3; keratin 4,keratin 7, keratin 15, keratin 18, tubulin-beta 3; keratin 4, keratin 7,keratin 15, keratin 18, keratin 19; keratin 4, keratin 8, keratin 15,keratin 19, tubulin-beta 3; keratin 4, keratin 8, keratin 15, keratin18, tubulin-beta 3; keratin 4, keratin 8, keratin 15, keratin 18,keratin 19; keratin 7, keratin 8, keratin 15, keratin 19, tubulin-beta3; keratin 7, keratin 8, keratin 15, keratin 18, tubulin-beta 3; andkeratin 7, keratin 8, keratin 15, keratin 18, keratin 19. Any marker setcan be used in combination with PSA. In addition, any of the above setsmay be modified to replace one or more markers in the marker set withone or more of the following additional markers: prostate-specificmembrane antigen (PSM), prostate stem cell antigen (PSCA), TMPRSS2,PDEF, prostate-specific gene-1 (HPG-1), PCA3, PCGEM1, and the genecluster P704P, P712P, and P775P. Any marker set can be used incombination with patient age.

In another embodiment, the invention contemplates marker sets with atleast seven (7) members, wherein one member is filamin A and theadditional six members may include, but are not limited to the followingsets: keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, andtubulin-beta 3; keratin 4, keratin 8, keratin 15, keratin 18, keratin19, and tubulin-beta 3; keratin 4, keratin 7, keratin 15, keratin 18,keratin 19, tubulin-beta 3; keratin 4, keratin 7, keratin 8, keratin 18,keratin 19, tubulin-beta 3; keratin 4, keratin 7, keratin 8, keratin 15,keratin 19, tubulin-beta 3; keratin 4, keratin 7, keratin 8, keratin 15,keratin 18, tubulin-beta 3; keratin 4, keratin 7, keratin 8, keratin 15,keratin 18, keratin 19; LY9, keratin 8, keratin 15, keratin 18, keratin19, tubulin-beta 3; LY9, keratin 7, keratin 15, keratin 18, keratin 19,tubulin-beta 3; LY9, keratin 7, keratin 8, keratin 18, keratin 19,tubulin-beta 3; LY9, keratin 7, keratin 8, keratin 15, keratin 19,tubulin-beta 3; LY9, keratin 7, keratin 8, keratin 15, keratin 18,tubulin-beta 3; LY9, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19; LY9, keratin 4, keratin 15, keratin 18, keratin 19,tubulin-beta 3; LY9, keratin 4, keratin 8, keratin 18, keratin 19,tubulin-beta 3; LY9, keratin 4, keratin 8, keratin 15, keratin 19,tubulin-beta 3; LY9, keratin 4, keratin 8, keratin 15, keratin 18,tubulin-beta 3; LY9, keratin 4, keratin 8, keratin 15, keratin 18,keratin 19; LY9, keratin 4, keratin 7, keratin 18, keratin 19, andtubulin-beta 3; LY9, keratin 4, keratin 7, keratin 15, keratin 19, andtubulin-beta 3; LY9, keratin 4, keratin 7, keratin 15, keratin 18,tubulin-beta 3; LY9, keratin 4, keratin 7, keratin 15, keratin 18,keratin 19; LY9, keratin 4, keratin 7, keratin 8, keratin 19,tubulin-beta 3; LY9, keratin 4, keratin 7, keratin 8, keratin 18,tubulin-beta 3; LY9, keratin 4, keratin 7, keratin 8, keratin 18,keratin 19; LY9, keratin 4, keratin 7, keratin 8, keratin 15,tubulin-beta 3; LY9, keratin 4, keratin 7, keratin 8, keratin 15,keratin 19; and LY9, keratin 4, keratin 7, keratin 8, keratin 15,keratin 18. Any marker set can be used in combination with PSA. Inaddition, any of the above sets may be modified to replace one or moremarkers in the marker set with one or more of the following additionalmarkers: prostate-specific membrane antigen (PSM), prostate stem cellantigen (PSCA), TMPRSS2, PDEF, prostate-specific gene-1 (HPG-1), PCA3,PCGEM1, and the gene cluster P704P, P712P, and P775P. Any maker set canbe used in combination with patient age.

In another embodiment, the invention contemplates marker sets with atleast eight (8) members, wherein one member is filamin A and theadditional seven members may include, but are not limited to thefollowing sets: keratin 4, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, tubulin-beta 3; LY9, keratin 7, keratin 8, keratin 15,keratin 18, keratin 19, tubulin-beta 3; LY9, keratin 4, keratin 8,keratin 15, keratin 18, keratin 19, tubulin-beta 3; LY9, keratin 4,keratin 7, keratin 15, keratin 18, keratin 19, tubulin-beta 3; LY9,keratin 4, keratin 7, keratin 8, keratin 18, keratin 19, tubulin-beta 3;LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 19,tubulin-beta 3; LY9, keratin 4, keratin 7, keratin 8, keratin 15,keratin 18, tubulin-beta 3; LY9, keratin 4, keratin 7, keratin 8,keratin 15, keratin 18, keratin 19; filamin B, keratin 7, keratin 8,keratin 15, keratin 18, keratin 19, tubulin-beta 3; filamin B, keratin4, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3;filamin B, keratin 4, keratin 7, keratin 15, keratin 18, keratin 19,tubulin-beta 3; filamin B, keratin 4, keratin 7, keratin 8, keratin 18,keratin 19, tubulin-beta 3; filamin B, keratin 4, keratin 7, keratin 8,keratin 15, keratin 19, tubulin-beta 3; filamin B, keratin 4, keratin 7,keratin 8, keratin 15, keratin 18, tubulin-beta 3; filamin B, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19; filamin B,LY9, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3;filamin B, LY9, keratin 7, keratin 15, keratin 18, keratin 19,tubulin-beta 3; filamin B, LY9, keratin 7, keratin 8, keratin 18,keratin 19, tubulin-beta 3; filamin B, LY9, keratin 7, keratin 8,keratin 15, keratin 19, tubulin-beta 3; filamin B, LY9, keratin 7,keratin 8, keratin 15, keratin 18, tubulin-beta 3; filamin B, LY9,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19; filamin B,LY9, keratin 4, keratin 15, keratin 18, keratin 19, tubulin-beta 3;filamin B, LY9, keratin 4, keratin 8, keratin 18, keratin 19,tubulin-beta 3; filamin B, LY9, keratin 4, keratin 8, keratin 15,keratin 19, tubulin-beta 3; filamin B, LY9, keratin 4, keratin 8,keratin 15, keratin 18, tubulin-beta 3; filamin B, LY9, keratin 4,keratin 8, keratin 15, keratin 18, keratin 19; filamin B, LY9, keratin4, keratin 7, keratin 18, keratin 19, and tubulin-beta 3; filamin B,LY9, keratin 4, keratin 7, keratin 15, keratin 19, and tubulin-beta 3;filamin B, LY9, keratin 4, keratin 7, keratin 15, keratin 18,tubulin-beta 3; filamin B, LY9, keratin 4, keratin 7, keratin 15,keratin 18, keratin 19; filamin B, LY9, keratin 4, keratin 7, keratin 8,keratin 19, tubulin-beta 3; filamin B, LY9, keratin 4, keratin 7,keratin 8, keratin 18, tubulin-beta 3; filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 18, keratin 19; filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, tubulin-beta 3; filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 19; and filamin B,LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18. Any markerset can be used in combination with PSA. In addition, any of the abovesets may be modified to replace one or more markers in the marker setwith one or more of the following additional markers: prostate-specificmembrane antigen (PSM), prostate stem cell antigen (PSCA), TMPRSS2,PDEF, prostate-specific gene-1 (HPG-1), PCA3, PCGEM1, and the genecluster P704P, P712P, and P775P. Any marker set can be used incombination with patient age.

In another embodiment, the invention contemplates marker sets with atleast nine (9) members, wherein one member is filamin A and theadditional eight members may include, but are not limited to thefollowing sets: LY9, keratin 4, keratin 7, keratin 8, keratin 15,keratin 18, keratin 19, tubulin-beta 3; filamin B, keratin 4, keratin 7,keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3; filaminB, LY9, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3; filamin B, LY9, keratin 4, keratin 8, keratin 15,keratin 18, keratin 19, tubulin-beta 3; filamin B, LY9, keratin 4,keratin 7, keratin 15, keratin 18, keratin 19, tubulin-beta 3; filaminB, LY9, keratin 4, keratin 7, keratin 8, keratin 18, keratin 19,tubulin-beta 3; filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin15, keratin 19, tubulin-beta 3; filamin B, LY9, keratin 4, keratin 7,keratin 8, keratin 15, keratin 18, tubulin-beta 3; and filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19. Anymarker set can be used in combination with PSA. In addition, any of theabove sets may be modified to replace one or more markers in the markerset with one or more of the following additional markers:prostate-specific membrane antigen (PSM), prostate stem cell antigen(PSCA), TMPRSS2, PDEF, prostate-specific gene-1 (HPG-1), PCA3, PCGEM1,and the gene cluster P704P, P712P, and P775P. Any marker set can be usedin combination with patient age.

In another embodiment, the invention contemplates marker sets with atleast ten (10) members, wherein one member is filamin A and theadditional nine members may include, but are not limited to thefollowing sets: filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin15, keratin 18, keratin 19, and tubulin-beta 3. In addition, any of theabove sets may be modified to replace one or more markers in the markerset with one or more of the following additional markers:prostate-specific membrane antigen (PSM), prostate stem cell antigen(PSCA), TMPRSS2, PDEF, prostate-specific gene-1 (HPG-1), PCA3, PCGEM1,and the gene cluster P704P, P712P, and P775P. Any marker set can be usedin combination with patient age.

Any marker set can be used in combination with PSA.

The invention provides for the use of various combinations andsub-combinations of markers. It is understood that any single marker orcombination of the markers provided herein can be used in the inventionunless clearly indicated otherwise. Further, any single marker orcombination of the markers of the invention can be used in conjunctionwith patient age. Alternatively, any single marker or combination of themarkers of the invention can be used in conjunction with PSA.Alternatively, any single marker or combination of the markers of theinvention can be used in conjunction with patient age and PSA.

Throughout the application, one or more of filamin B, LY9 and keratin 19is understood as any of: filamin B; LY9; keratin 19; filamin B and LY9;filamin B and keratin 19; LY9 and keratin 19; or filamin B, LY9, andkeratin 19. Further, any single marker or combination of the markers ofthe invention can be used in conjunction with PSA. Further, any singlemarker or combination of the markers of the invention can be used inconjunction with patient age. Preferably, each marker set includes, inaddition if not already indicated, filamin A.

Throughout the application, combination of the filamin B and LY9 withPSA is understood as any of filamin B; LY9; filamin B and PSA; filamin Band LY9; LY9 and PSA; filamin B, LY9, and PSA. Preferably, each markerset includes, in addition if not already indicated, filamin A.Preferably, each marker set includes, in addition if not alreadyindicated, patient age.

Throughout the application, one or more prostate cancer markers selectedfrom the group consisting of keratin 4, keratin 7, keratin 8, keratin15, keratin 18, and tubulin beta-3 is understood as any of keratin 4;keratin 7; keratin 8; keratin 15; keratin 18; tubulin beta-3; keratin 4and keratin 7; keratin 4 and keratin 8; keratin 4 and keratin 15;keratin 4 and keratin 18; keratin 4 and tubulin beta-3; keratin 7 andkeratin 8; keratin 7 and keratin 15; keratin 7 and keratin 18; keratinand tubulin beta-3; keratin 8 and keratin 15; keratin 8 and keratin 18;keratin 8 and tubulin beta-3; keratin 15 and keratin 18; keratin 15 andtubulin beta-3; keratin 18 and tubulin beta-3; keratin 4, keratin 7 andkeratin 8; keratin 4, keratin 7 and keratin 15; keratin 4, keratin 7 andkeratin 18; keratin 4, keratin 7 and tubulin beta-3; keratin 4, keratin8 and keratin 15; keratin 4, keratin 8 and keratin 18; keratin 4,keratin 8 and tubulin beta-e; keratin 4, keratin 15 and keratin 18;keratin 4, keratin 15 and tubulin beta-e; keratin 4, keratin 18 andtubulin beta-3; kertin 4, keratin 7, keratin 8 and keratin 15; keratin4, keratin 7, keratin 8 and keratin 18; keratin 4, keratin 7, keratin 8and tubulin beta-3; keratin 4, keratin 8, keratin 15 and keratin 18;keratin 4, keratin 8, keratin 15 and tubulin beta-3; keratin 4, keratin15, keratin 18 and tubulin beta-3; keratin 4, keratin 7, keratin 8,keratin 15 and keratin 18; keratin 4, keratin 7, keratin 8, keratin 15,and tubulin beta-3; keratin 4, keratin 7, keratin 8, keratin 18, andtubulin beta-3; keratin 4, keratin 7, keratin 15, keratin 18, andtubulin beta-3; keratin 4, keratin 8, keratin 15, keratin 18, andtubulin beta-3; or keratin 7, keratin 8, keratin 15, keratin 18, andtubulin beta-3. Further, any single marker or combination of the markersof the invention can be used in conjunction with PSA. Preferably, eachmarker set includes, in addition if not already indicated, filamin A.Preferably, each marker set includes, in addition if not alreadyindicated, patient age.

Throughout the application, one or more prostate cancer markers selectedfrom the group consisting of keratin 7, 15, and 19 is understood as anyof keratin 7; keratin 15; keratin 19; keratin 7 and 15; keratin 7 and19; keratin 15 and 19; and keratin 7, 15, and 19. Further, any singlemarker or combination of the markers of the invention can be used inconjunction with PSA. Preferably, each marker set includes, in additionif not already indicated, filamin A. Preferably, each marker setincludes, in addition if not already indicated, patient age.

Throughout the application, one or more prostate cancer markers selectedfrom the group consisting of keratin 7, 8, and 15 is understood as anyof keratin 7; keratin 8; keratin 15; keratin 7 and 8; keratin 7 and 15;keratin 8 and 15; and keratin 7, 8, and 15. Further, any single markeror combination of the markers of the invention can be used inconjunction with PSA. Preferably, each marker set includes, in additionif not already indicated, filamin A. Preferably, each marker setincludes, in addition if not already indicated, patient age.

Throughout the application, one or more prostate cancer markers selectedfrom the group consisting of keratin 7 and 15 is understood as any ofkeratin 7; keratin 15; or keratin 7 and 15. Further, any single markeror combination of the markers of the invention can be used inconjunction with PSA. Preferably, each marker set includes, in additionif not already indicated, filamin A. Preferably, each marker setincludes, in addition if not already indicated, patient age.

Throughout the application, one or more prostate cancer markers selectedfrom the group consisting filamin B, LY9, or keratin 19 is understood asany of filamin B; LY9; keratin 19; filamin B and LY9; filamin B andkeratin 19; LY9, and keratin 19; and filamin B, LY9, and keratin 19.Further, any single marker or combination of the markers of theinvention can be used in conjunction with PSA. Preferably, each markerset includes, in addition if not already indicated, filamin A.Preferably, each marker set includes, in addition if not alreadyindicated, patient age.

In another aspect, the present invention provides for the identificationof a “diagnostic signature” or “disease profile” based on the levels ofthe biomarkers of the invention in a biological sample, including in adiseased tissue (e.g., prostate tumor) or directly from the serum orblood, that correlates with the presence and/or risk and/or prognosis ofprostate cancer. The “levels of the biomarkers” can refer to theexpression level of the biomarker genes, e.g., by measuring theexpression levels of the biomarker mRNAs. The “levels of the biomarkers”can also refer to level of biomarker polypeptides in a biologicalsample, e.g., prostate tissue or serum. The collection or totality ofexpression levels of biomarker polypeptides and/or nucleic acidmolecules provide a diagnostic signature that correlates with thepresence and/or diagnosis and/or progression of prostate cancer. Thebiomarkers for obtaining a diagnostic signature or disease profile ofthe invention are meant to encompass any measurable characteristic thatreflects in a quantitative or qualitative manner the physiological stateof an organism, e.g, whether the organism has prostate cancer. Thephysiological state of an organism is inclusive of any disease ornon-disease state, e.g., a subject having prostate cancer or a subjectwho is otherwise healthy. Said another way, the biomarkers used foridentifying a diagnostic signature or disease profile of the inventioninclude characteristics that can be objectively measured and evaluatedas indicators of normal processes, pathogenic processes, orpharmacologic responses to a therapeutic intervention, including, inparticular, prostate cancer. Biomarkers can be clinical parameters(e.g., age, performance status), laboratory measures (e.g., molecularbiomarkers, such as prostate specific antigen), imaging-based measures,or genetic or other molecular determinants Examples of biomarkersinclude, for example, polypeptides, peptides, polypeptide fragments,proteins, antibodies, hormones, polynucleotides, RNA or RNA fragments,microRNA (miRNAs), lipids, polysaccharides, and other bodily metabolitesthat are diagnostic and/or indicative and/or predictive of anoncological disease, e.g., prostate cancer. Examples of biomarkers alsoinclude polypeptides, peptides, polypeptide fragments, proteins,antibodies, hormones, polynucleotides, RNA or RNA fragments, microRNA(miRNAs), lipids, polysaccharides, and other bodily metabolites whichare diagnostic and/or indicative and/or predictive of any stage orclinical phase of an oncological disease, e.g., Gleason grade 1, grade2, grade 3, grade 4, or grade 5 prostate cancer.

In a particular embodiment, a prostate cancer disease profile ordiagnostic signature is determined on the basis of the combination offilamin A together with one or more additional biomarkers of prostatecancer, which can include, but are not limited to prostate specificantigen (PSA), filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin15, keratin 18, keratin 19, and tubulin-beta 3, as well as additionalmarkers PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1. Other markersthat may be used in combination with filamin A include any measurablecharacteristic that reflects in a quantitative or qualitative manner thephysiological state of an organism, e.g, whether the organism hasprostate cancer. Such characteristics may include patient age. Thephysiological state of an organism is inclusive of any disease ornon-disease state, e.g., a subject having prostate cancer or a subjectwho is otherwise healthy. Said another way, the biomarkers of theinvention that may be used in combination with filamin A includecharacteristics that can be objectively measured and evaluated asindicators of normal processes, pathogenic processes, or pharmacologicresponses to a therapeutic intervention, including, in particular,prostate cancer. Such combination biomarkers can be clinical parameters(e.g., age, performance status), laboratory measures (e.g., molecularbiomarkers, such as prostate specific antigen), imaging-based measures,or genetic or other molecular determinants Example of biomarkers for usein combination with filamin A include, for example, polypeptides,peptides, polypeptide fragments, proteins, antibodies, hormones,polynucleotides, RNA or RNA fragments, microRNA (miRNAs), lipids,polysaccharides, and other bodily metabolites that are diagnostic and/orindicative and/or predictive of prostate cancer, or any particular stageor phase of prostate cancer, e.g., Gleason grade 1, grade 2, grade 3,grade 4, or grade 5 prostate cancer. In certain embodiments, biomarkersfor use in combination with filamin A include polypeptides, peptides,polypeptide fragments, proteins, antibodies, hormones, polynucleotides,RNA or RNA fragments, microRNA (miRNAs), lipids, polysaccharides, andother bodily metabolites which are diagnostic and/or indicative and/orpredictive of prostate cancer, or any stage or clinical phase thereof,e.g., Gleason grade 1, grade 2, grade 3, grade 4, or grade 5 prostatecancer or TNM classifications. In other embodiments, the presentinvention also involves the analysis and consideration of any clinicaland/or patient-related health data, for example, data obtained from anElectronic Medical Record (e.g., collection of electronic healthinformation about individual patients or populations relating to varioustypes of data, such as, demographics, medical history, medication andallergies, immunization status, laboratory test results, radiologyimages, vital signs, personal statistics like age and weight, andbilling information).

In certain embodiments, the diagnostic signature is obtained by (1)detecting the level of filamin A in a biological sample, (2) detectingthe level(s) of one or more additional biomarkers that may include, butare not limited to prostate specific antigen (PSA), filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1, and(3) comparing the levels of filamin A and the one or more additionalbiomarkers to the levels of the same biomarkers from a control sample,(4) determining if the filamin A and the one or more additionalbiomarkers detected in the biological sample are above a certainthreshold level. If filamin A and at least 1 additionally detectedbiomarker is above the threshold level, then the diagnostic signature isindicative of prostate cancer in the biological sample. In certainembodiments, the diagnostic signature can be determined based on analgorithm or computer program that predicts whether the biologicalsample is cancerous based on the levels of filamin A and the one or moreadditional biomarkers.

In certain other embodiments, the diagnostic signature is obtained by(1) detecting the level of filamin A in a biological sample, (2)detecting the level(s) of two or more additional biomarkers that mayinclude, but are not limited to prostate specific antigen (PSA), filaminB, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1,and (3) comparing the levels of filamin A and the two or more additionalbiomarkers to the levels of the same biomarkers from a control sample,(4) determining if the filamin A and the two or more additionalbiomarkers detected in the biological sample are above a certainthreshold level. If filamin A and at least 2 additionally detectedbiomarkers are above the threshold level, then the diagnostic signatureis indicative of prostate cancer in the biological sample. In certainembodiments, the diagnostic signature can be determined based on analgorithm or computer program that predicts whether the biologicalsample is cancerous based on the levels of filamin A and the two or moreadditional biomarkers. In other embodiments, the diagnostic signaturealso takes into account the patient's age.

In certain other embodiments, the diagnostic signature is obtained by(1) detecting the level of filamin A in a biological sample, (2)detecting the level(s) of three or more additional biomarkers that mayinclude, but are not limited to prostate specific antigen (PSA), filaminB, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1,and (3) comparing the levels of filamin A and the three or moreadditional biomarkers to the levels of the same biomarkers from acontrol sample, (4) determining if the filamin A and the three or moreadditional biomarkers detected in the biological sample are above acertain threshold level. If filamin A and at least 3 additionallydetected biomarkers are above the threshold level, then the diagnosticsignature is indicative of prostate cancer in the biological sample. Incertain embodiments, the diagnostic signature can be determined based onan algorithm or computer program that predicts whether the biologicalsample is cancerous based on the levels of filamin A and the three ormore additional biomarkers. In other embodiments, the diagnosticsignature also takes into account the patient's age.

In other embodiments, the diagnostic signature is obtained by (1)detecting the level of filamin A in a biological sample, (2) detectingthe level(s) of four or more additional biomarkers that may include, butare not limited to prostate specific antigen (PSA), filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1, and(3) comparing the levels of filamin A and the four or more additionalbiomarkers to the levels of the same biomarkers from a control sample,(4) determining if the filamin A and the four or more additionalbiomarkers detected in the biological sample are above a certainthreshold level. If filamin A and at least 4 additionally detectedbiomarkers are above the threshold level, then the diagnostic signatureis indicative of prostate cancer in the biological sample. In certainembodiments, the diagnostic signature can be determined based on analgorithm or computer program that predicts whether the biologicalsample is cancerous based on the levels of filamin A and the four ormore additional biomarkers. In other embodiments, the diagnosticsignature also takes into account the patient's age.

In other embodiments, the diagnostic signature is obtained by (1)detecting the level of filamin A in a biological sample, (2) detectingthe level(s) of five or more additional biomarkers that may include, butare not limited to prostate specific antigen (PSA), filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1, and(3) comparing the levels of filamin A and the five or more additionalbiomarkers to the levels of the same biomarkers from a control sample,(4) determining if the filamin A and the five or more additionalbiomarkers detected in the biological sample are above a certainthreshold level. If filamin A and at least 5 additionally detectedbiomarkers are above the threshold level, then the diagnostic signatureis indicative of prostate cancer in the biological sample. In certainembodiments, the diagnostic signature can be determined based on analgorithm or computer program that predicts whether the biologicalsample is cancerous based on the levels of filamin A and the five ormore additional biomarkers. In other embodiments, the diagnosticsignature also takes into account the patient's age.

In other embodiments, the diagnostic signature is obtained by (1)detecting the level of filamin A in a biological sample, (2) detectingthe level(s) of six or more additional biomarkers that may include, butare not limited to prostate specific antigen (PSA), filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1, and(3) comparing the levels of filamin A and the six or more additionalbiomarkers to the levels of the same biomarkers from a control sample,(4) determining if the filamin A and the six or more additionalbiomarkers detected in the biological sample are above a certainthreshold level. If filamin A and at least 6 additionally detectedbiomarkers are above the threshold level, then the diagnostic signatureis indicative of prostate cancer in the biological sample. In certainembodiments, the diagnostic signature can be determined based on analgorithm or computer program that predicts whether the biologicalsample is cancerous based on the levels of filamin A and the six or moreadditional biomarkers. In other embodiments, the diagnostic signaturealso takes into account the patient's age.

In other embodiments, the diagnostic signature is obtained by (1)detecting the level of filamin A in a biological sample, (2) detectingthe level(s) of seven or more additional biomarkers that may include,but are not limited to prostate specific antigen (PSA), filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1, and(3) comparing the levels of filamin A and the seven or more additionalbiomarkers to the levels of the same biomarkers from a control sample,(4) determining if the filamin A and the seven or more additionalbiomarkers detected in the biological sample are above a certainthreshold level. If filamin A and at least 7 additionally detectedbiomarkers are above the threshold level, then the diagnostic signatureis indicative of prostate cancer in the biological sample. In certainembodiments, the diagnostic signature can be determined based on analgorithm or computer program that predicts whether the biologicalsample is cancerous based on the levels of filamin A and the seven ormore additional biomarkers. In other embodiments, the diagnosticsignature also takes into account the patient's age.

In other embodiments, the diagnostic signature is obtained by (1)detecting the level of filamin A in a biological sample, (2) detectingthe level(s) of eight or more additional biomarkers that may include,but are not limited to prostate specific antigen (PSA), filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1, and(3) comparing the levels of filamin A and the eight or more additionalbiomarkers to the levels of the same biomarkers from a control sample,(4) determining if the filamin A and the eight or more additionalbiomarkers detected in the biological sample are above a certainthreshold level. If filamin A and at least 8 additionally detectedbiomarkers are above the threshold level, then the diagnostic signatureis indicative of prostate cancer in the biological sample. In certainembodiments, the diagnostic signature can be determined based on analgorithm or computer program that predicts whether the biologicalsample is cancerous based on the levels of filamin A and the eight ormore additional biomarkers. In other embodiments, the diagnosticsignature also takes into account the patient's age.

In other embodiments, the diagnostic signature is obtained by (1)detecting the level of filamin A in a biological sample, (2) detectingthe level(s) of nine or more additional biomarkers that may include, butare not limited to prostate specific antigen (PSA), filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1, and(3) comparing the levels of filamin A and the nine or more additionalbiomarkers to the levels of the same biomarkers from a control sample,(4) determining if the filamin A and the nine or more additionalbiomarkers detected in the biological sample are above a certainthreshold level. If filamin A and at least 9 additionally detectedbiomarkers are above the threshold level, then the diagnostic signatureis indicative of prostate cancer in the biological sample. In certainembodiments, the diagnostic signature can be determined based on analgorithm or computer program that predicts whether the biologicalsample is cancerous based on the levels of filamin A and the nine ormore additional biomarkers. In other embodiments, the diagnosticsignature also takes into account the patient's age.

In accordance with various embodiments, algorithms may be employed topredict whether or not a biological sample is likely to be diseased,e.g., have prostate cancer. The skilled artisan will appreciate that analgorithm can be any computation, formula, statistical survey, nomogram,look-up table, decision tree method, or computer program which processesa set of input variables (e.g., number of markers (n) which have beendetected at a level exceeding some threshold level, or number of markers(n) which have been detected at a level below some threshold level)through a number of well-defined successive steps to eventually producea score or “output,” e.g., a diagnosis of prostate cancer. Any suitablealgorithm—whether computer-based or manual-based (e.g., look-uptable)—is contemplated herein.

In certain embodiments, an algorithm of the invention used to predictwhether a biological sample has prostate cancer producing a score on thebasis of the detecte level of filamin A in the sample and the level(s)at least one, or two, or three, or four, or five, or six, or seven, oreight, or nine or more additional prostate cancer markers (e.g.,selected from the group consisting of prostate specific antigen (PSA),filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, andPCGEM1), wherein if the score is above a certain threshold score, thenthe biological sample has prostate cancer. In certain embodiments, thealgorithm also produces a score using the patient's age as a continuouspredictor variable. For example, increased age is associated with higherrisk of prostate cancer.

In certain embodiments, an algorithm of the invention used to predictwhether a biological sample has prostate cancer producing a score on thebasis of the detecte level of filamin A in the sample and the level(s)at least one, or two, or three, or four, or five, or six, or seven, oreight, or nine or more additional prostate cancer markers (e.g.,selected from the group consisting of prostate specific antigen (PSA),filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, andPCGEM1), wherein if the score is below a certain threshold score, thenthe biological sample has prostate cancer. In certain embodiments, thealgorithm also produces a score using the patient's age as a continuouspredictor variable.

In certain embodiments, an algorithm of the invention used to predictwhether a biological sample has prostate cancer producing a score on thebasis of the detecte level of filamin A in the sample and the level(s)at least one, or two, or three, or four, or five, or six, or seven, oreight, or nine or more additional prostate cancer markers (e.g.,selected from the group consisting of prostate specific antigen (PSA),filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, andPCGEM1), wherein if the score is above a certain threshold score, thenthe biological sample does not have prostate cancer. In certainembodiments, the algorithm also produces a score using the patient's ageas a continuous predictor variable.

In certain embodiments, an algorithm of the invention used to predictwhether a biological sample has prostate cancer producing a score on thebasis of the detecte level of filamin A in the sample and the level(s)at least one, or two, or three, or four, or five, or six, or seven, oreight, or nine or more additional prostate cancer markers (e.g.,selected from the group consisting of prostate specific antigen (PSA),filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, andPCGEM1), wherein if the score is below a certain threshold score, thenthe biological sample does not have prostate cancer. In certainembodiments, the algorithm also produces a score using the patient's ageas a continuous predictor variable.

Moreover, a prostate cancer disease profile or signature may be obtainedby detecting filamin A in combination with at least one other biomarker,or more preferably, with at least two other biomarkers, or still morepreferably, with at least three other biomarkers, or even morepreferably with at least four other biomarkers. Still further, filamin Ain certain embodiments, may be used in combination with at least fiveother markers, or at least six other biomarkers, or at least seven otherbiomarkers, or at least eight other biomarkers, or at least nine otherbiomarkers, or at least ten other biomarkers, or at least eleven otherbiomarkers, or at least twelve other biomarkers, or at least thirteenother biomarkers, or at least fourteen other biomarkers, or at leastfifteen other biomarkers, or at least sixteen other biomarkers, or atleast seventeen other biomarkers, or at least eighteen other biomarkers,or at least nineteen other biomarkers, or at least twenty otherbiomarkers. Further still, filamin A may be used in combination with amultitude of other biomarkers, including, for example, with betweenabout 20-50 other biomarkers, or between 50-100, or between 100-500, orbetween 500-1000, or between 1000-10,000 or biomarkers or more. Incertain embodiments, the patient's age is also used as a continuouspredictor variable. For example, increased age is associated withincreased risk of prostate cancer diagnosis.

In certain embodiments, the biomarkers of the invention can includevariant sequences. More particularly, the binding agents/reagents usedfor detecting the biomarkers of the invention can bind and/or identifyvariants of the biomarkers of the invention. As used herein, the term“variant” comprehends nucleotide or amino acid sequences different fromthe specifically identified sequences, wherein one or more nucleotidesor amino acid residues is deleted, substituted, or added. Variants maybe naturally occurring allelic variants, or non-naturally occurringvariants. Variant sequences (polynucleotide or polypeptide) preferablyexhibit at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to asequence disclosed herein. The percentage identity is determined byaligning the two sequences to be compared as described below,determining the number of identical residues in the aligned portion,dividing that number by the total number of residues in the inventive(queried) sequence, and multiplying the result by 100.

In addition to exhibiting the recited level of sequence identity,variants of the disclosed polypeptide biomarkers are preferablythemselves expressed in subjects with prostate cancer at levels that arehigher or lower than the levels of expression in normal, healthyindividuals.

Variant sequences generally differ from the specifically identifiedsequence only by conservative substitutions, deletions or modifications.As used herein, a “conservative substitution” is one in which an aminoacid is substituted for another amino acid that has similar properties,such that one skilled in the art of peptide chemistry would expect thesecondary structure and hydropathic nature of the polypeptide to besubstantially unchanged. In general, the following groups of amino acidsrepresent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn,ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4)lys, arg, his; and (5) phe, tyr, trp, his. Variants may also, oralternatively, contain other modifications, including the deletion oraddition of amino acids that have minimal influence on the antigenicproperties, secondary structure and hydropathic nature of thepolypeptide. For example, a polypeptide may be conjugated to a signal(or leader) sequence at the N-terminal end of the protein whichco-translationally or post-translationally directs transfer of theprotein. The polypeptide may also be conjugated to a linker or othersequence for ease of synthesis, purification or identification of thepolypeptide (e.g., poly-His), or to enhance binding of the polypeptideto a solid support. For example, a polypeptide may be conjugated to animmunoglobulin Fc region.

Polypeptide and polynucleotide sequences may be aligned, and percentagesof identical amino acids or nucleotides in a specified region may bedetermined against another polypeptide or polynucleotide sequence, usingcomputer algorithms that are publicly available. The percentage identityof a polynucleotide or polypeptide sequence is determined by aligningpolynucleotide and polypeptide sequences using appropriate algorithms,such as BLASTN or BLASTP, respectively, set to default parameters;identifying the number of identical nucleic or amino acids over thealigned portions; dividing the number of identical nucleic or aminoacids by the total number of nucleic or amino acids of thepolynucleotide or polypeptide of the present invention; and thenmultiplying by 100 to determine the percentage identity.

Two exemplary algorithms for aligning and identifying the identity ofpolynucleotide sequences are the BLASTN and FASTA algorithms. Thealignment and identity of polypeptide sequences may be examined usingthe BLASTP algorithm. BLASTX and FASTX algorithms compare nucleotidequery sequences translated in all reading frames against polypeptidesequences. The FASTA and FASTX algorithms are described in Pearson andLipman, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988; and in Pearson,Methods in Enzymol. 183:63-98, 1990. The FASTA software package isavailable from the University of Virginia, Charlottesville, Va.22906-9025. The FASTA algorithm, set to the default parameters describedin the documentation and distributed with the algorithm, may be used inthe determination of polynucleotide variants. The readme files for FASTAand FASTX Version 2.0x that are distributed with the algorithms describethe use of the algorithms and describe the default parameters.

The BLASTN software is available on the NCBI anonymous FTP server and isavailable from the National Center for Biotechnology Information (NCBI),National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md.20894. The BLASTN algorithm Version 2.0.6 [Sep. 10, 1998] and Version2.0.11 [Jan. 20, 2000] set to the default parameters described in thedocumentation and distributed with the algorithm, is preferred for usein the determination of variants according to the present invention. Theuse of the BLAST family of algorithms, including BLASTN, is described atNCBI's website and in the publication of Altschul, et al., “Gapped BLASTand PSI-BLAST: a new generation of protein database search programs,”Nucleic Acids Res. 25:3389-3402, 1997.

In an alternative embodiment, variant polypeptides are encoded bypolynucleotide sequences that hybridize to a disclosed polynucleotideunder stringent conditions. Stringent hybridization conditions fordetermining complementarity include salt conditions of less than about 1M, more usually less than about 500 mM, and preferably less than about200 mM. Hybridization temperatures can be as low as 5° C., but aregenerally greater than about 22° C., more preferably greater than about30° C., and most preferably greater than about 37° C. Longer DNAfragments may require higher hybridization temperatures for specifichybridization. Since the stringency of hybridization may be affected byother factors such as probe composition, presence of organic solventsand extent of base mismatching, the combination of parameters is moreimportant than the absolute measure of any one alone. An example of“stringent conditions” is prewashing in a solution of 6×SSC, 0.2% SDS;hybridizing at 65° C., 6×SSC, 0.2% SDS overnight; followed by two washesof 30 minutes each in 1×SSC, 0.1% SDS at 65° C. and two washes of 30minutes each in 0.2×SSC, 0.1% SDS at 65° C.

D. Tissue Samples

The present invention may be practiced with any suitable biologicalsample that potentially contains, expresses, includes, a detectabledisease biomarker, e.g., a polypeptide biomarker, a nucleic acidbiomarkers, a mRNA biomarker, a microRNA biomarker. For example, thebiological sample may be obtained from sources that include whole bloodand serum to diseased and/or healthy tissue, for example, biopsy ofprostate tumor. The methods of the invention may especially be appliedto the study of any prostate tissue sample, i.e., a sample of prostatetissue or fluid, as well as cells (or their progeny) isolated from suchtissue or fluid. In another embodiment, the present invention may bepracticed with any suitable Prostate tissue samples which are freshlyisolated or which have been frozen or stored after having been collectedfrom a subject, or archival tissue samples, for example, with knowndiagnosis, treatment and/or outcome history. Prostate tissue may becollected by any non-invasive means, such as, for example, fine needleaspiration and needle biopsy, or alternatively, by an invasive method,including, for example, surgical biopsy.

The inventive methods may be performed at the single cell level (e.g.,isolation and testing of cancerous cells from the prostate tissuesample). However, preferably, the inventive methods are performed usinga sample comprising many cells, where the assay is “averaging”expression over the entire collection of cells and tissue present in thesample. Preferably, there is enough of the prostate tissue sample toaccurately and reliably determine the expression levels of the set ofgenes of interest. In certain embodiments, multiple samples may be takenfrom the same prostate tissue in order to obtain a representativesampling of the tissue. In addition, sufficient biological material canbe obtained in order to perform duplicate, triplicate or further roundsof testing.

Any commercial device or system for isolating and/or obtaining prostatetissue and/or blood or other biological products, and/or for processingsaid materials prior to conducting a detection reaction is contemplated.

In certain embodiments, the present invention relates to detectingbiomarker nucleic acid molecules (e.g., mRNA encoding filamin A, antigen(PSA), filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin 15,kertin 18, keratin 19, and tubulin-beta 3). In such embodiments, RNA canbe extracted from a biological sample, e.g., a prostate tissue sample,before analysis. Methods of RNA extraction are well known in the art(see, for example, J. Sambrook et al., “Molecular Cloning: A LaboratoryManual”, 1989, 2^(nd) Ed., Cold Spring Harbour Laboratory Press: NewYork). Most methods of RNA isolation from bodily fluids or tissues arebased on the disruption of the tissue in the presence of proteindenaturants to quickly and effectively inactivate RNases. Generally, RNAisolation reagents comprise, among other components, guanidiniumthiocyanate and/or beta-mercaptoethanol, which are known to act as RNaseinhibitors. Isolated total RNA is then further purified from the proteincontaminants and concentrated by selective ethanol precipitations,phenol/chloroform extractions followed by isopropanol precipitation(see, for example, P. Chomczynski and N. Sacchi, Anal. Biochem., 1987,162: 156-159) or cesium chloride, lithium chloride or cesiumtrifluoroacetate gradient centrifugations.

Numerous different and versatile kits can be used to extract RNA (i.e.,total RNA or mRNA) from bodily fluids or tissues (e.g., prostate tissuesamples) and are commercially available from, for example, Ambion, Inc.(Austin, Tex.), Amersham Biosciences (Piscataway, N.J.), BD BiosciencesClontech (Palo Alto, Calif.), BioRad Laboratories (Hercules, Calif.),GIBCO BRL (Gaithersburg, Md.), and Giagen, Inc. (Valencia, Calif.). UserGuides that describe in great detail the protocol to be followed areusually included in all these kits. Sensitivity, processing time andcost may be different from one kit to another. One of ordinary skill inthe art can easily select the kit(s) most appropriate for a particularsituation.

In certain embodiments, after extraction, mRNA is amplified, andtranscribed into cDNA, which can then serve as template for multiplerounds of transcription by the appropriate RNA polymerase. Amplificationmethods are well known in the art (see, for example, A. R. Kimmel and S.L. Berger, Methods Enzymol. 1987, 152: 307-316; J. Sambrook et al.,“Molecular Cloning: A Laboratory Manual”, 1989, 2.sup.nd Ed., ColdSpring Harbour Laboratory Press: New York; “Short Protocols in MolecularBiology”, F. M. Ausubel (Ed.), 2002, 5.sup.th Ed., John Wiley & Sons;U.S. Pat. Nos. 4,683,195; 4,683,202 and 4,800,159). Reversetranscription reactions may be carried out using non-specific primers,such as an anchored oligo-dT primer, or random sequence primers, orusing a target-specific primer complementary to the RNA for each geneticprobe being monitored, or using thermostable DNA polymerases (such asavian myeloblastosis virus reverse transcriptase or Moloney murineleukemia virus reverse transcriptase).

In certain embodiments, the RNA isolated from the prostate tissue sample(for example, after amplification and/or conversion to cDNA or cRNA) islabeled with a detectable agent before being analyzed. The role of adetectable agent is to facilitate detection of RNA or to allowvisualization of hybridized nucleic acid fragments (e.g., nucleic acidfragments hybridized to genetic probes in an array-based assay).Preferably, the detectable agent is selected such that it generates asignal which can be measured and whose intensity is related to theamount of labeled nucleic acids present in the sample being analyzed. Inarray-based analysis methods, the detectable agent is also preferablyselected such that it generates a localized signal, thereby allowingspatial resolution of the signal from each spot on the array.

Methods for labeling nucleic acid molecules are well-known in the art.For a review of labeling protocols, label detection techniques andrecent developments in the field, see, for example, L. J. Kricka, Ann.Clin. Biochem. 2002, 39: 114-129; R. P. van Gijlswijk et al., ExpertRev. Mol. Diagn. 2001, 1: 81-91; and S. Joos et al., J. Biotechnol.1994, 35: 135-153. Standard nucleic acid labeling methods include:incorporation of radioactive agents, direct attachment of fluorescentdyes (see, for example, L. M. Smith et al., Nucl. Acids Res. 1985, 13:2399-2412) or of enzymes (see, for example, B. A. Connoly and P. Rider,Nucl. Acids. Res. 1985, 13: 4485-4502); chemical modifications ofnucleic acid fragments making them detectable immunochemically or byother affinity reactions (see, for example, T. R. Broker et al., Nucl.Acids Res. 1978, 5: 363-384; E. A. Bayer et al., Methods of Biochem.Analysis, 1980, 26: 1-45; R. Langer et al., Proc. Natl. Acad. Sci. USA,1981, 78: 6633-6637; R. W. Richardson et al., Nucl. Acids Res. 1983, 11:6167-6184; D. J. Brigati et al., Virol. 1983, 126: 32-50; P. Tchen etal., Proc. Natl Acad. Sci. USA, 1984, 81: 3466-3470; J. E. Landegent etal., Exp. Cell Res. 1984, 15: 61-72; and A. H. Hopman et al., Exp. CellRes. 1987, 169: 357-368); and enzyme-mediated labeling methods, such asrandom priming, nick translation, PCR and tailing with terminaltransferase (for a review on enzymatic labeling, see, for example, J.Temsamani and S. Agrawal, Mol. Biotechnol. 1996, 5: 223-232).

Any of a wide variety of detectable agents can be used in the practiceof the present invention. Suitable detectable agents include, but arenot limited to: various ligands, radionuclides, fluorescent dyes,chemiluminescent agents, microparticles (such as, for example, quantumdots, nanocrystals, phosphors and the like), enzymes (such as, forexample, those used in an ELISA, i.e., horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase), colorimetriclabels, magnetic labels, and biotin, dioxigenin or other haptens andproteins for which antisera or monoclonal antibodies are available.

However, in some embodiments, the expression levels are determined bydetecting the expression of a gene product (e.g., protein) therebyeliminating the need to obtain a genetic sample (e.g., RNA) from theprostate tissue sample.

In still other embodiments, the present invention relates to preparing aprediction model for prostate and/or the likelihood of relapse ofprostate cancer by preparing a model for prostate cancer based onmeasuring the biomarkers of the invention in known control samples. Moreparticularly, the present invention relates in some embodiments topreparing a predictive model by evaluating the biomarkers of theinvention, e.g., filamin A in combination with one or more of prostatespecific antigen (PSA), filamin B, LY9, keratin 4, keratin 7, keratin 8,keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2,PDEF, HPG-1, PCA3, and PCGEM1.

The skilled person will appreciate that patient tissue samplescontaining prostate cells or prostate cancer cells may be used in themethods of the present invention including, but not limited to thoseaimed at predicting relapse probability. In these embodiments, the levelof expression of the signature gene can be assessed by assessing theamount, e.g. absolute amount or concentration, of a signature geneproduct, e.g., protein and RNA transcript encoded by the signature geneand fragments of the protein and RNA transcript) in a sample, e.g.,stool and/or blood obtained from a patient. The sample can, of course,be subjected to a variety of well-known post-collection preparative andstorage techniques (e.g. fixation, storage, freezing, lysis,homogenization, DNA or RNA extraction, ultrafiltration, concentration,evaporation, centrifugation, etc.) prior to assessing the amount of thesignature gene product in the sample.

The invention further relates to the preparation of a model for prostatecancer or prostate cancer relapse by evaluating the biomarkers of theinvention in known samples of prostate cancer. More particularly, thepresent invention relates to a prostate cancer model for diagnosingand/or monitoring and/or prognosing prostate cancer or prostate cancerrelapse using the biomarkers of the invention, which can include filaminA and at least one other prostate cancer related marker selected fromthe group consisting of filamin B, LY9, keratin 4, keratin 7, keratin 8,keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSA, PSM, PSCA,TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1.

In the methods of the invention aimed at preparing a model for prostatecancer and/or prostate cancer relapse prediction, it is understood thatthe particular clinical outcome associated with each sample contributingto the model preferably should be known. Consequently, the model can beestablished using archived tissue samples. In the methods of theinvention aimed at preparing a model for prostate cancer and/or prostatecancer relapse prediction, total RNA can be generally extracted from thesource material of interest, generally an archived tissue such as aformalin-fixed, paraffin-embedded tissue, and subsequently purified.Methods for obtaining robust and reproducible gene expression patternsfrom archived tissues, including formalin-fixed, paraffin-embedded(FFPE) tissues are taught in U.S. Publ. No. 2004/0259105, which isincorporated herein by reference in its entirety. Commercial kits andprotocols for RNA extraction from FFPE tissues are available including,for example, ROCHE High Pure RNA Paraffin Kit (Roche) MasterPure™Complete DNA and RNA Purification Kit (EPICENTRE®Madison, Wis.);Paraffin Block RNA Isolation Kit (Ambion, Inc.) and RNeasy™ Mini kit(Qiagen, Chatsworth, Calif.).

The use of FFPE tissues as a source of RNA for RT-PCR has been describedpreviously (Stanta et al., Biotechniques 11:304-308 (1991); Stanta etal., Methods Mol. Biol. 86:23-26 (1998); Jackson et al., Lancet 1:1391(1989); Jackson et al., J. Clin. Pathol. 43:499-504 (1999); Finke etal., Biotechniques 14:448-453 (1993); Goldsworthy et al., Mol. Carcinog.25:86-91 (1999); Stanta and Bonin, Biotechniques 24:271-276 (1998);Godfrey et al., J. Mol. Diagnostics 2:84 (2000); Specht et al., J. Mol.Med. 78:B27 (2000); Specht et al., Am. J. Pathol. 158:419-429 (2001)).For quick analysis of the RNA quality, RT-PCR can be performed utilizinga pair of primers targeting a short fragment in a highly expressed gene,for example, actin, ubiquitin, gapdh or other well-described commonlyused housekeeping gene. If the cDNA synthesized from the RNA sample canbe amplified using this pair of primers, then the sample is suitable forthe a quantitative measurements of RNA target sequences by any methodpreferred, for example, the DASL assay, which requires only a short cDNAfragment for the annealing of query oligonucleotides.

There are numerous tissue banks and collections including exhaustivesamples from all stages of a wide variety of disease states, mostnotably cancer and in particular, prostate cancer. The ability toperform genotyping and/or gene expression analysis, including bothqualitative and quantitative analysis on these samples enables theapplication of this methodology to the methods of the invention. Inparticular, the ability to establish a correlation of gene expressionand a known predictor of disease extent and/or outcome by probing thegenetic state of tissue samples for which clinical outcome is alreadyknown, allows for the establishment of a correlation between aparticular molecular signature and the known predictor, such as aGleason score, to derive a score that allows for a more sensitiveprognosis than that based on the known predictor alone. The skilledperson will appreciate that by building databases of molecularsignatures from tissue samples of known outcomes, many such correlationscan be established, thus allowing both diagnosis and prognosis of anycondition. Thus, such approaches may be used to correlate the expressionlevels of the biomarkers of the invention, e.g., filamin A and at leastone other prostate cancer related marker selected from the groupconsisting of filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin15, keratin 18, keratin 19, tubulin-beta 3 and PSA, to a particularstage of prostate cancer.

Tissue samples useful for preparing a model for prostate cancerprediction include, for example, paraffin and polymer embedded samples,ethanol embedded samples and/or formalin and formaldehyde embeddedtissues, although any suitable sample may be used. In general, nucleicacids isolated from archived samples can be highly degraded and thequality of nucleic preparation can depend on several factors, includingthe sample shelf life, fixation technique and isolation method. However,using the methodologies taught in U.S. Publ. No. 2004/0259105, whichhave the significant advantage that short or degraded targets can beused for analysis as long as the sequence is long enough to hybridizewith the oligonucleotide probes, highly reproducible results can beobtained that closely mimic results found in fresh samples.

Archived tissue samples, which can be used for all methods of theinvention, typically have been obtained from a source and preserved.Preferred methods of preservation include, but are not limited toparaffin embedding, ethanol fixation and formalin, includingformaldehyde and other derivatives, fixation as are known in the art. Atissue sample may be temporally “old”, e.g. months or years old, orrecently fixed. For example, post-surgical procedures generally includea fixation step on excised tissue for histological analysis. In apreferred embodiment, the tissue sample is a diseased tissue sample,particularly a prostate cancer tissue, including primary and secondarytumor tissues as well as lymph node tissue and metastatic tissue.

Thus, an archived sample can be heterogeneous and encompass more thanone cell or tissue type, for example, tumor and non-tumor tissue.Preferred tissue samples include solid tumor samples including, but notlimited to, tumors of the prostate. It is understood that inapplications of the present invention to conditions other than prostatecancer, the tumor source can be brain, bone, heart, breast, ovaries,prostate, uterus, spleen, pancreas, liver, kidneys, bladder, stomach andmuscle. Similarly, depending on the condition, suitable tissue samplesinclude, but are not limited to, bodily fluids (including, but notlimited to, blood, urine, serum, lymph, saliva, anal and vaginalsecretions, perspiration and semen, of virtually any organism, withmammalian samples being preferred and human samples being particularlypreferred). In embodiments directed to methods of establishing a modelfor prostate cancer relapse prediction, the tissue sample is one forwhich patient history and outcome is known. Generally, the inventionmethods can be practiced with the signature gene sequence contained inan archived sample or can be practiced with signature gene sequencesthat have been physically separated from the sample prior to performinga method of the invention.

E. Detection and/or Measurement of Biomarkers

The present invention contemplates any suitable means, techniques,and/or procedures for detecting and/or measuring the biomarkers of theinvention. The skilled artisan will appreciate that the methodologiesemployed to measure the biomarkers of the invention will depend at leaston the type of biomarker being detected or measured (e.g., mRNAbiomarker or polypeptide biomarker) and the source of the biologicalsample (e.g., whole blood versus prostate biopsy tissue). Certainbiological sample may also require certain specialized treatments priorto measuring the biomarkers of the invention, e.g., the preparation ofmRNA from a biopsy tissue in the case where mRNA biomarkers are beingmeasured.

1. Detection of Nucleic Acid Biomarkers

In certain embodiments, the invention involves the detection of nucleicacid biomarkers, e.g., mRNA biomarkers of filamin A alone or filamin Ain combination with at least one other prostate cancer related markerselected from the group consisting of filamin B, LY9, keratin 4, keratin7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSA,PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1.

In various embodiments, the diagnostic/prognostic methods of the presentinvention generally involve the determination of expression levels of aset of genes in a prostate tissue sample. Determination of geneexpression levels in the practice of the inventive methods may beperformed by any suitable method. For example, determination of geneexpression levels may be performed by detecting the expression of mRNAexpressed from the genes of interest and/or by detecting the expressionof a polypeptide encoded by the genes.

For detecting nucleic acids encoding biomarkers of the invention, anysuitable method can be used, including, but not limited to, Southernblot analysis, Northern blot analysis, polymerase chain reaction (PCR)(see, for example, U.S. Pat. Nos. 4,683,195; 4,683,202, and 6,040,166;“PCR Protocols: A Guide to Methods and Applications”, Innis et al.(Eds), 1990, Academic Press: New York), reverse transcriptase PCR(RT-PCT), anchored PCR, competitive PCR (see, for example, U.S. Pat. No.5,747,251), rapid amplification of cDNA ends (RACE) (see, for example,“Gene Cloning and Analysis: Current Innovations, 1997, pp. 99-115);ligase chain reaction (LCR) (see, for example, EP 01 320 308), one-sidedPCR (Ohara et al., Proc. Natl. Acad. Sci., 1989, 86: 5673-5677), in situhybridization, Taqman-based assays (Holland et al., Proc. Natl. Acad.Sci., 1991, 88: 7276-7280), differential display (see, for example,Liang et al., Nucl. Acid. Res., 1993, 21: 3269-3275) and other RNAfingerprinting techniques, nucleic acid sequence based amplification(NASBA) and other transcription based amplification systems (see, forexample, U.S. Pat. Nos. 5,409,818 and 5,554,527), Qbeta Replicase,Strand Displacement Amplification (SDA), Repair Chain Reaction (RCR),nuclease protection assays, subtraction-based methods, Rapid-Scan®, etc.

In other embodiments, gene expression levels of biomarkers of interestmay be determined by amplifying complementary DNA (cDNA) orcomplementary RNA (cRNA) produced from mRNA and analyzing it using amicroarray. A number of different array configurations and methods oftheir production are known to those skilled in the art (see, forexample, U.S. Pat. Nos. 5,445,934; 5,532,128; 5,556,752; 5,242,974;5,384,261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327;5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554,501; 5,561,071;5,571,639; 5,593,839; 5,599,695; 5,624,711; 5,658,734; and 5,700,637).Microarray technology allows for the measurement of the steady-statemRNA level of a large number of genes simultaneously. Microarrayscurrently in wide use include cDNA arrays and oligonucleotide arrays.Analyses using microarrays are generally based on measurements of theintensity of the signal received from a labeled probe used to detect acDNA sequence from the sample that hybridizes to a nucleic acid probeimmobilized at a known location on the microarray (see, for example,U.S. Pat. Nos. 6,004,755; 6,218,114; 6,218,122; and 6,271,002).Array-based gene expression methods are known in the art and have beendescribed in numerous scientific publications as well as in patents(see, for example, M. Schena et al., Science, 1995, 270: 467-470; M.Schena et al., Proc. Natl. Acad. Sci. USA 1996, 93: 10614-10619; J. J.Chen et al., Genomics, 1998, 51: 313-324; U.S. Pat. Nos. 5,143,854;5,445,934; 5,807,522; 5,837,832; 6,040,138; 6,045,996; 6,284,460; and6,607,885).

In one particular embodiment, the invention comprises a method foridentification of prostate cancer cells in a biological sample byamplifying and detecting nucleic acids corresponding to the novelprostate cancer biomarkers, and or panels of biomarkers that includefilamin A alone or filamin A in combination with one or more markersselected from the group consisting of filamin B, LY9, keratin 4, keratin7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSA,PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1. The biological samplemay be any tissue or fluid in which prostate cancer cells might bepresent. Various embodiments include radical prostatectomy specimens,pathological specimens, bone marrow aspirate, bone marrow biopsy, lymphnode aspirate, lymph node biopsy, spleen tissue, fine needle aspirate,skin biopsy or organ tissue biopsy. Other embodiments include sampleswhere the body fluid is peripheral blood, serum, plasma, lymph fluid,ascites, serous fluid, pleural effusion, sputum, cerebrospinal fluid,lacrimal fluid, stool, prostatic fluid or urine.

Nucleic acid used as a template for amplification can be isolated fromcells contained in the biological sample, according to standardmethodologies. (Sambrook et al., 1989) The nucleic acid may be genomicDNA or fractionated or whole cell RNA. Where RNA is used, it may bedesired to convert the RNA to a complementary cDNA. In one embodiment,the RNA is whole cell RNA and is used directly as the template foramplification.

Pairs of primers that selectively hybridize to nucleic acidscorresponding to any of the prostate cancer biomarker nucleotidesequences identified herein are contacted with the isolated nucleic acidunder conditions that permit selective hybridization. Once hybridized,the nucleic acid:primer complex is contacted with one or more enzymesthat facilitate template-dependent nucleic acid synthesis. Multiplerounds of amplification, also referred to as “cycles,” are conducteduntil a sufficient amount of amplification product is produced. Next,the amplification product is detected. In certain applications, thedetection may be performed by visual means. Alternatively, the detectionmay involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of incorporated radiolabelor fluorescent label or even via a system using electrical or thermalimpulse signals (Affymax technology; Bellus, 1994). Following detection,one may compare the results seen in a given patient with a statisticallysignificant reference group of normal patients and prostate, cancerpatients. In this way, it is possible to correlate the amount of nucleicacid detected with various clinical states.

The term primer, as defined herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty base pairs in length, but longer sequences may beemployed. Primers may be provided in double-stranded or single-strandedform, although the single-stranded form is preferred.

A number of template dependent processes are available to amplify thenucleic acid sequences present in a given template sample. One of thebest known amplification methods is the polymerase chain reaction(referred to as PCR) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1990, each ofwhich is incorporated herein by reference in its entirety.

In PCR, two primer sequences are prepared which are complementary toregions on opposite complementary strands of the target nucleic acidsequence. An excess of deoxynucleoside triphosphates are added to areaction mixture along with a DNA polymerase, e.g., Taq polymerase. Ifthe target nucleic acid sequence is present in a sample, the primerswill bind to the target nucleic acid and the polymerase will cause theprimers to be extended along the target nucleic acid sequence by addingon nucleotides. By raising and lowering the temperature of the reactionmixture, the extended primers will dissociate from the target nucleicacid to form reaction products, excess primers will bind to the targetnucleic acid and to the reaction products and the process is repeated.

A reverse transcriptase PCR amplification procedure may be performed inorder to quantify the amount of mRNA amplified. Methods of reversetranscribing RNA into cDNA are well known and described in Sambrook etal., 1989. Alternative methods for reverse transcription utilizethermostable DNA polymerases. These methods are described in WO 90/07641filed Dec. 21, 1990. Polymerase chain reaction methodologies are wellknown in the art.

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in European Application No. 320 308, incorporated herein byreference in its entirely. In LCR, two complementary probe pairs areprepared, and in the presence of the target sequence, each pair willbind to opposite complementary strands of the target such that theyabut. In the presence of a ligase, the two probe pairs will link to forma single unit. By temperature cycling, as in PCR, bound ligated unitsdissociate from the target and then serve as “target sequences” forligation of excess probe pairs. U.S. Pat. No. 4,883,750 describes amethod similar to LCR for binding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, alsomay be used as still another amplification method in the presentinvention. In this method, a replicative sequence of RNA which has aregion complementary to that of a target is added to a sample in thepresence of an RNA polymerase. The polymerase will copy the replicativesequence which may then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[α-thio]triphosphates in one strand of arestriction site also may be useful in the amplification of nucleicacids in the present invention. Walker et al. (1992), incorporatedherein by reference in its entirety.

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e., nick translation. Asimilar method, called Repair Chain Reaction (RCR), involves annealingseveral probes throughout a region targeted for amplification, followedby a repair reaction in which only two of the four bases are present.The other two bases may be added as biotinylated derivatives for easydetection. A similar approach is used in SDA. Target specific sequencesalso may be detected using a cyclic probe reaction (CPR). In CPR, aprobe having 3′ and 5′ sequences of non-specific DNA and a middlesequence of specific RNA is hybridized to DNA which is present in asample. Upon hybridization, the reaction is treated with RNase H, andthe products of the probe identified as distinctive products which arereleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated.

Still other amplification methods described in GB Application No. 2 202328, and in PCT Application No. PCT/US89/01025, each of which isincorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,“modified” primers are used in a PCR like, template and enzyme dependentsynthesis. The primers may be modified by labeling with a capture moiety(e.g., biotin) and/or a detector moiety (e.g., enzyme). In the latterapplication, an excess of labeled probes are added to a sample. In thepresence of the target sequence, the probe binds and is cleavedcatalytically. After cleavage, the target sequence is released intact tobe bound by excess probe. Cleavage of the labeled probe signals thepresence of the target sequence.

Other contemplated nucleic acid amplification procedures includetranscription-based amplification systems (TAS), including nucleic acidsequence based amplification (NASBA) and 3SR. Kwoh et al. (1989);Gingeras et al., PCT Application WO 88/10315, incorporated herein byreference in their entirety. In NASBA, the nucleic acids may be preparedfor amplification by standard phenol/chloroform extraction, heatdenaturation of a clinical sample, treatment with lysis buffer andminispin columns for isolation of DNA and RNA or guanidinium chlorideextraction of RNA. These amplification techniques involve annealing aprimer which has target specific sequences. Following polymerization,DNA/RNA hybrids are digested with RNase H while double stranded DNAmolecules are heat denatured again. In either case the single strandedDNA is made fully double stranded by addition of second target specificprimer, followed by polymerization. The double-stranded DNA moleculesare then multiply transcribed by a polymerase such as T7 or SP6. In anisothermal cyclic reaction, the RNA's are reverse transcribed intodouble stranded DNA, and transcribed once against with a polymerase suchas T7 or SP6. The resulting products, whether truncated or complete,indicate target specific sequences.

Davey et al., European Application No. 329 822 (incorporated herein byreference in its entirely) disclose a nucleic acid amplification processinvolving cyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA,and double-stranded DNA (dsDNA), which may be used in accordance withthe present invention. The ssRNA is a first template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H(RNase H, anRNase specific for RNA in duplex with either DNA or RNA). The resultantssDNA is a second template for a second primer, which also includes thesequences of an RNA polymerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase 1), resulting in a double-stranded DNA(“dsDNA”) molecule, having a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence may be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies may thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification may be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence may be chosen to be in the form ofeither DNA or RNA.

Miller et al., PCT Application WO 89/06700 (incorporated herein byreference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic, i.e., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “race” and “one-sidedPCR™.” Frohman (1990) and Ohara et al. (1989), each herein incorporatedby reference in their entirety.

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, alsomay be used in the amplification step of the present invention. Wu etal. (1989), incorporated herein by reference in its entirety.

Oligonucleotide probes or primers of the present invention may be of anysuitable length, depending on the particular assay format and theparticular needs and targeted sequences employed. In a preferredembodiment, the oligonucleotide probes or primers are at least 10nucleotides in length (preferably, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 . . . ) and theymay be adapted to be especially suited for a chosen nucleic acidamplification system and/or hybridization system used. Longer probes andprimers are also within the scope of the present invention as well knownin the art. Primers having more than 30, more than 40, more than 50nucleotides and probes having more than 100, more than 200, more than300, more than 500 more than 800 and more than 1000 nucleotides inlength are also covered by the present invention. Of course, longerprimers have the disadvantage of being more expensive and thus, primershaving between 12 and 30 nucleotides in length are usually designed andused in the art. As well known in the art, probes ranging from 10 tomore than 2000 nucleotides in length can be used in the methods of thepresent invention. As for the % of identity described above,non-specifically described sizes of probes and primers (e.g., 16, 17,31, 24, 39, 350, 450, 550, 900, 1240 nucleotides, . . . ) are alsowithin the scope of the present invention. In one embodiment, theoligonucleotide probes or primers of the present invention specificallyhybridize with a filamin A RNA (or its complementary sequence) or afilamin A mRNA. More preferably, the filamin A primers and probes willbe chosen to detect a filamin A RNA which is associated with prostatecancer.

In other embodiments, the detection means can utilize a hybridizationtechnique, e.g., where a specific primer or probe is selected to annealto a target biomarker of interest, e.g., filamin A, and thereafterdetection of selective hybridization is made. As commonly known in theart, the oligonucleotide probes and primers can be designed by takinginto consideration the melting point of hybridization thereof with itstargeted sequence (see below and in Sambrook et al., 1989, MolecularCloning—A Laboratory Manual, 2nd Edition, CSH Laboratories; Ausubel etal., 1994, in Current Protocols in Molecular Biology, John Wiley & SonsInc., N.Y.).

To enable hybridization to occur under the assay conditions of thepresent invention, oligonucleotide primers and probes should comprise anoligonucleotide sequence that has at least 70% (at least 71%, 72%, 73%,74%), preferably at least 75% (75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%) and more preferably at least 90%(90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%) identity to aportion of a filamin A or polynucleotide of another biomarker of theinvention. Probes and primers of the present invention are those thathybridize under stringent hybridization conditions and those thathybridize to biomarker homologs of the invention under at leastmoderately stringent conditions. In certain embodiments probes andprimers of the present invention have complete sequence identity to thebiomarkers of the invention (filamin A, gene sequences (e.g., cDNA ormRNA). It should be understood that other probes and primers could beeasily designed and used in the present invention based on thebiomarkers of the invention disclosed herein by using methods ofcomputer alignment and sequence analysis known in the art (cf. MolecularCloning: A Laboratory Manual, Third Edition, edited by Cold SpringHarbor Laboratory, 2000).

2. Detection of Polypeptide Biomarkers

The present invention contemplates any suitable method for detectingpolypeptide biomarkers of the invention. In certain embodiments, thedetection method is an immunodetection method involving an antibody thatspecifically binds to one or more of the biomarkers of the inventioninvention, e.g., filamin A alone or filamin A in combination with atleast one other prostate cancer related marker selected from the groupconsisting of filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin15, keratin 18, keratin 19, tubulin-beta 3, PSA, PSM, PSCA, TMPRSS2,PDEF, HPG-1, PCA3, and PCGEM1. The steps of various usefulimmunodetection methods have been described in the scientificliterature, such as, e.g., Nakamura et al. (1987), which is incorporatedherein by reference.

In general, the immunobinding methods include obtaining a samplesuspected of containing a biomarker protein, peptide or antibody, andcontacting the sample with an antibody or protein or peptide inaccordance with the present invention, as the case may be, underconditions effective to allow the formation of immunocomplexes.

The immunobinding methods include methods for detecting or quantifyingthe amount of a reactive component in a sample, which methods requirethe detection or quantitation of any immune complexes formed during thebinding process. Here, one would obtain a sample suspected of containinga prostate specific protein, peptide or a corresponding antibody, andcontact the sample with an antibody or encoded protein or peptide, asthe case may be, and then detect or quantify the amount of immunecomplexes formed under the specific conditions.

In terms of biomarker detection, the biological sample analyzed may beany sample that is suspected of containing a prostate cancer-specificbiomarker, such as, filamin A and at least one other prostate cancerrelated marker selected from the group consisting of filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3 and PSA. The biological sample may be, for example, aprostate or lymph node tissue section or specimen, a homogenized tissueextract, an isolated cell, a cell membrane preparation, separated orpurified forms of any of the above protein-containing compositions, oreven any biological fluid that comes into contact with prostate tissues,including blood or lymphatic fluid.

Contacting the chosen biological sample with the protein (e.g., filaminA or antigen thereof to bind with a anti-filamin A antibody in theblood), peptide (e.g., filamin A fragment that binds with a anti-filaminA antibody in the blood), or antibody (e.g., as a detection reagent thatbinds filamin A in a biological sample) under conditions effective andfor a period of time sufficient to allow the formation of immunecomplexes (primary immune complexes). Generally, complex formation is amatter of simply adding the composition to the biological sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, any antigenspresent. After this time, the sample-antibody composition, such as atissue section, ELISA plate, dot blot or Western blot, will generally bewashed to remove any non-specifically bound antibody species, allowingonly those antibodies specifically bound within the primary immunecomplexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any radioactive, fluorescent, biological orenzymatic tags or labels of standard use in the art. U.S. patentsconcerning the use of such labels include U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241,each incorporated herein by reference. Of course, one may findadditional advantages through the use of a secondary binding ligand suchas a second antibody or a biotin/avidin ligand binding arrangement, asis known in the art.

The encoded protein (e.g., filamin A), peptide (e.g., filamin A peptide)or corresponding antibody (anti-filamin A antibody as detection reagent)employed in the detection may itself be linked to a detectable label,wherein one would then simply detect this label, thereby allowing theamount of the primary immune complexes in the composition to bedetermined.

Alternatively, the first added component that becomes bound within theprimary immune complexes may be detected by means of a second bindingligand that has binding affinity for the encoded protein, peptide orcorresponding antibody. In these cases, the second binding ligand may belinked to a detectable label. The second binding ligand is itself oftenan antibody, which may thus be termed a “secondary” antibody. Theprimary immune complexes are contacted with the labeled, secondarybinding ligand, or antibody, under conditions effective and for a periodof time sufficient to allow the formation of secondary immune complexes.The secondary immune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the encoded protein, peptide or correspondingantibody is used to form secondary immune complexes, as described above.After washing, the secondary immune complexes are contacted with a thirdbinding ligand or antibody that has binding affinity for the secondantibody, again under conditions effective and for a period of timesufficient to allow the formation of immune complexes (tertiary immunecomplexes). The third ligand or antibody is linked to a detectablelabel, allowing detection of the tertiary immune complexes thus formed.This system may provide for signal amplification if this is desired.

The immunodetection methods of the present invention have evidentutility in the diagnosis of conditions such as prostate cancer. Here, abiological or clinical sample suspected of containing either the encodedprotein or peptide or corresponding antibody is used. However, theseembodiments also have applications to non-clinical samples, such as inthe tittering of antigen or antibody samples, in the selection ofhybridomas, and the like.

The present invention, in particular, contemplates the use of ELISAs asa type of immunodetection assay. It is contemplated that the biomarkerproteins or peptides of the invention will find utility as immunogens inELISA assays in diagnosis and prognostic monitoring of prostate cancer.Immunoassays, in their most simple and direct sense, are binding assays.Certain preferred immunoassays are the various types of enzyme linkedimmunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in theart. Immunohistochemical detection using tissue sections is alsoparticularly useful. However, it will be readily appreciated thatdetection is not limited to such techniques, and Western blotting, dotblotting, FACS analyses, and the like also may be used.

In one exemplary ELISA, antibodies binding to the biomarkers of theinvention are immobilized onto a selected surface exhibiting proteinaffinity, such as a well in a polystyrene microtiter plate. Then, a testcomposition suspected of containing the prostate cancer marker antigen,such as a clinical sample, is added to the wells. After binding andwashing to remove non-specifically bound immunecomplexes, the boundantigen may be detected. Detection is generally achieved by the additionof a second antibody specific for the target protein, that is linked toa detectable label. This type of ELISA is a simple “sandwich ELISA.”Detection also may be achieved by the addition of a second antibody,followed by the addition of a third antibody that has binding affinityfor the second antibody, with the third antibody being linked to adetectable label.

In another exemplary ELISA, the samples suspected of containing theprostate cancer marker antigen are immobilized onto the well surface andthen contacted with the anti-biomarker antibodies of the invention.After binding and washing to remove non-specifically boundimmunecomplexes, the bound antigen is detected. Where the initialantibodies are linked to a detectable label, the immunecomplexes may bedetected directly. Again, the immunecomplexes may be detected using asecond antibody that has binding affinity for the first antibody, withthe second antibody being linked to a detectable label.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating or binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes.These are described as follows.

In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period of hours. The wellsof the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein and solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the control human prostate,cancer and/or clinical or biological sample to be tested underconditions effective to allow immunecomplex (antigen/antibody)formation. Detection of the immunecomplex then requires a labeledsecondary binding ligand or antibody, or a secondary binding ligand orantibody in conjunction with a labeled tertiary antibody or thirdbinding ligand.

The phrase “under conditions effective to allow immunecomplex(antigen/antibody) formation” means that the conditions preferablyinclude diluting the antigens and antibodies with solutions such as BSA,bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween.These added agents also tend to assist in the reduction of nonspecificbackground.

The “suitable” conditions also mean that the incubation is at atemperature and for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 h, attemperatures preferably on the order of 25 to 27° C., or may beovernight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. A preferred washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immunecomplexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immunecomplexes may bedetermined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. Preferably, this will be an enzymethat will generate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact andincubate the first or second immunecomplex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immunecomplex formation (e.g., incubation for 2 h at roomtemperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea and bromocresolpurple. Quantitation is then achieved by measuring the degree of colorgeneration, e.g., using a visible spectra spectrophotometer.

The protein biomarkers of the invention (e.g., filamin A alone or incombination with any one or more of filamin B, LY9, keratin 4, keratin7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSA,PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1) can also be measured,quantitated, detected, and otherwise analyzed using protein massspectrometry methods and instrumentation. Protein mass spectrometryrefers to the application of mass spectrometry to the study of proteins.Although not intending to be limiting, two approaches are typically usedfor characterizing proteins using mass spectrometry. In the first,intact proteins are ionized and then introduced to a mass analyzer. Thisapproach is referred to as “top-down” strategy of protein analysis. Thetwo primary methods for ionization of whole proteins are electrosprayionization (ESI) and matrix-assisted laser desorption/ionization(MALDI). In the second approach, proteins are enzymatically digestedinto smaller peptides using a protease such as trypsin. Subsequentlythese peptides are introduced into the mass spectrometer and identifiedby peptide mass fingerprinting or tandem mass spectrometry. Hence, thislatter approach (also called “bottom-up” proteomics) uses identificationat the peptide level to infer the existence of proteins.

Whole protein mass analysis of the biomarkers of the invention can beconducted using time-of-flight (TOF) MS, or Fourier transform ioncyclotron resonance (FT-ICR). These two types of instruments are usefulbecause of their wide mass range, and in the case of FT-ICR, its highmass accuracy. The most widely used instruments for peptide massanalysis are the MALDI time-of-flight instruments as they permit theacquisition of peptide mass fingerprints (PMFs) at high pace (1 PMF canbe analyzed in approx. 10 sec). Multiple stage quadrupole-time-of-flightand the quadrupole ion trap also find use in this application.

The biomarkers of the invention can also be measured in complex mixturesof proteins and molecules that co-exist in a biological medium orsample, however, fractionation of the sample may be required and iscontemplated herein. It will be appreciated that ionization of complexmixtures of proteins can result in situation where the more abundantproteins have a tendency to “drown” or suppress signals from lessabundant proteins in the same sample. In addition, the mass spectrumfrom a complex mixture can be difficult to interpret because of theoverwhelming number of mixture components. Fractionation can be used tofirst separate any complex mixture of proteins prior to massspectrometry analysis. Two methods are widely used to fractionateproteins, or their peptide products from an enzymatic digestion. Thefirst method fractionates whole proteins and is called two-dimensionalgel electrophoresis. The second method, high performance liquidchromatography (LC or HPLC) is used to fractionate peptides afterenzymatic digestion. In some situations, it may be desirable to combineboth of these techniques. Any other suitable methods known in the artfor fractionating protein mixtures are also contemplated herein.

Gel spots identified on a 2D Gel are usually attributable to oneprotein. If the identity of the protein is desired, usually the methodof in-gel digestion is applied, where the protein spot of interest isexcised, and digested proteolytically. The peptide masses resulting fromthe digestion can be determined by mass spectrometry using peptide massfingerprinting. If this information does not allow unequivocalidentification of the protein, its peptides can be subject to tandemmass spectrometry for de novo sequencing.

Characterization of protein mixtures using HPLC/MS may also be referredto in the art as “shotgun proteomics” and MuDPIT (Multi-DimensionalProtein Identification Technology). A peptide mixture that results fromdigestion of a protein mixture is fractionated by one or two steps ofliquid chromatography (LC). The eluent from the chromatography stage canbe either directly introduced to the mass spectrometer throughelectrospray ionization, or laid down on a series of small spots forlater mass analysis using MALDI.

The biomarkers of the present invention (e.g., filamin A alone or incombination with any one or more of filamin B, LY9, keratin 4, keratin7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSA,PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1) can be identifiedusing MS using a variety of techniques, all of which are contemplatedherein. Peptide mass fingerprinting uses the masses of proteolyticpeptides as input to a search of a database of predicted masses thatwould arise from digestion of a list of known proteins. If a proteinsequence in the reference list gives rise to a significant number ofpredicted masses that match the experimental values, there is someevidence that this protein was present in the original sample. It willbe further appreciated that the development of methods andinstrumentation for automated, data-dependent electrospray ionization(ESI) tandem mass spectrometry (MS/MS) in conjunction withmicrocapillary liquid chromatography (LC) and database searching hassignificantly increased the sensitivity and speed of the identificationof gel-separated proteins. Microcapillary LC-MS/MS has been usedsuccessfully for the large-scale identification of individual proteinsdirectly from mixtures without gel electrophoretic separation (Link etal., 1999; Opitek et al., 1997).

Several recent methods allow for the quantitation of proteins by massspectrometry. For example, stable (e.g., non-radioactive) heavierisotopes of carbon (¹³C) or nitrogen (¹⁵N) can be incorporated into onesample while the other one can be labeled with corresponding lightisotopes (e.g. ¹²C and ¹⁴N). The two samples are mixed before theanalysis. Peptides derived from the different samples can bedistinguished due to their mass difference. The ratio of their peakintensities corresponds to the relative abundance ratio of the peptides(and proteins). The most popular methods for isotope labeling are SILAC(stable isotope labeling by amino acids in cell culture),trypsin-catalyzed ¹⁸O labeling, ICAT (isotope coded affinity tagging),iTRAQ (isobaric tags for relative and absolute quantitation).“Semi-quantitative” mass spectrometry can be performed without labelingof samples. Typically, this is done with MALDI analysis (in linearmode). The peak intensity, or the peak area, from individual molecules(typically proteins) is here correlated to the amount of protein in thesample. However, the individual signal depends on the primary structureof the protein, on the complexity of the sample, and on the settings ofthe instrument. Other types of “label-free” quantitative massspectrometry, uses the spectral counts (or peptide counts) of digestedproteins as a means for determining relative protein amounts.

In one embodiment, any one or more of the biomarkers of the invention(e.g., filamin A alone or in combination with any one or more of filaminB, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin19, tubulin-beta 3, PSA, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, andPCGEM1) can be identified and quantified from a complex biologicalsample using mass spectroscopy in accordance with the followingexemplary method, which is not intended to limit the invention or theuse of other mass spectrometry-based methods.

In the first step of this embodiment, (A) a biological sample, e.g., abiological sample suspected of having prostate cancer, which comprises acomplex mixture of protein (including at least one biomarker ofinterest) is fragmented and labeled with a stable isotope X. (B) Next, aknown amount of an internal standard is added to the biological sample,wherein the internal standard is prepared by fragmenting a standardprotein that is identical to the at least one target biomarker ofinterest, and labeled with a stable isotope Y. (C) This sample obtainedis then introduced in an LC-MS/MS device, and multiple reactionmonitoring (MRM) analysis is performed using MRM transitions selectedfor the internal standard to obtain an MRM chromatogram. (D) The MRMchromatogram is then viewed to identify a target peptide biomarkerderived from the biological sample that shows the same retention time asa peptide derived from the internal standard (an internal standardpeptide), and quantifying the target protein biomarker in the testsample by comparing the peak area of the internal standard peptide withthe peak area of the target peptide biomarker.

Any suitable biological sample may be used as a starting point forLC-MS/MS/MRM analysis, including biological samples derived blood,urine, saliva, hair, cells, cell tissues, biopsy materials, and treatedproducts thereof; and protein-containing samples prepared by generecombination techniques.

Each of the above steps (A) to (D) is described further below.

Step (A) (Fragmentation and Labeling). In step (A), the target proteinbiomarker is fragmented to a collection of peptides, which issubsequently labeled with a stable isotope X. To fragment the targetprotein, for example, methods of digesting the target protein with aproteolytic enzyme (protease) such as trypsin, and chemical cleavagemethods, such as a method using cyanogen bromide, can be used. Digestionby protease is preferable. It is known that a given mole quantity ofprotein produces the same mole quantity for each tryptic peptidecleavage product if the proteolytic digest is allowed to proceed tocompletion. Thus, determining the mole quantity of tryptic peptide to agiven protein allows determination of the mole quantity of the originalprotein in the sample. Absolute quantification of the target protein canbe accomplished by determining the absolute amount of the targetprotein-derived peptides contained in the protease digestion (collectionof peptides). Accordingly, in order to allow the proteolytic digest toproceed to completion, reduction and alkylation treatments arepreferably performed before protease digestion with trypsin to reduceand alkylate the disulfide bonds contained in the target protein.

Subsequently, the obtained digest (collection of peptides, comprisingpeptides of the target biomarker in the biological sample) is subjectedto labeling with a stable isotope X. Examples of stable isotopes Xinclude ¹H and ²H for hydrogen atoms, ¹²C and ¹³C for carbon atoms, and¹⁴N and ¹⁵N for nitrogen atoms. Any isotope can be suitably selectedtherefrom. Labeling by a stable isotope X can be performed by reactingthe digest (collection of peptides) with a reagent containing the stableisotope.

Preferable examples of such reagents that are commercially availableinclude mTRAQ (registered trademark) (produced by Applied Biosystems),which is an amine-specific stable isotope reagent kit. mTRAQ is composedof 2 or 3 types of reagents (mTRAQ-light and mTRAQ-heavy; or mTRAQ-DO,mTRAQ-D4, and mTRAQ-D8) that have a constant mass differencetherebetween as a result of isotope-labeling, and that are bound to theN-terminus of a peptide or the primary amine of a lysine residue.

Step (B) (Addition of the Internal Standard). In step (B), a knownamount of an internal standard is added to the sample obtained in step(A). The internal standard used herein is a digest (collection ofpeptides) obtained by fragmenting a protein (standard protein)consisting of the same amino acid sequence as the target protein (targetbiomarker) to be measured, and labeling the obtained digest (collectionof peptides) with a stable isotope Y. The fragmentation treatment can beperformed in the same manner as above for the target protein. Labelingwith a stable isotope Y can also be performed in the same manner asabove for the target protein. However, the stable isotope Y used hereinmust be an isotope that has a mass different from that of the stableisotope X used for labeling the target protein digest. For example, inthe case of using the aforementioned mTRAQ (registered trademark)(produced by Applied Biosystems), when mTRAQ-light is used to label atarget protein digest, mTRAQ-heavy should be used to label a standardprotein digest.

Step (C) (LC-MS/MS and MRM Analysis). In step (C), the sample obtainedin step (B) is first placed in an LC-MS/MS device, and then multiplereaction monitoring (MRM) analysis is performed using MRM transitionsselected for the internal standard. By LC (liquid chromatography) usingthe LC-MS/MS device, the sample (collection of peptides labeled with astable isotope) obtained in step (B) is separated first byone-dimensional or multi-dimensional high-performance liquidchromatography. Specific examples of such liquid chromatography includecation exchange chromatography, in which separation is conducted byutilizing electric charge difference between peptides; andreversed-phase chromatography, in which separation is conducted byutilizing hydrophobicity difference between peptides. Both of thesemethods may be used in combination.

Subsequently, each of the separated peptides is subjected to tandem massspectrometry by using a tandem mass spectrometer (MS/MS spectrometer)comprising two mass spectrometers connected in series. The use of such amass spectrometer enables the detection of several fmol levels of atarget protein. Furthermore, MS/MS analysis enables the analysis ofinternal sequence information on peptides, thus enabling identificationwithout false positives. Other types of MS analyzers may also be used,including magnetic sector mass spectrometers (Sector MS), quadrupolemass spectrometers (QMS), time-of-flight mass spectrometers (TOFMS), andFourier transform ion cyclotron resonance mass spectrometers (FT-ICRMS),and combinations of these analyzers.

Subsequently, the obtained data are put through a search engine toperform a spectral assignment and to list the peptides experimentallydetected for each protein. The detected peptides are preferably groupedfor each protein, and preferably at least three fragments having an m/zvalue larger than that of the precursor ion and at least three fragmentswith an m/z value of, preferably, 500 or more are selected from eachMS/MS spectrum in descending order of signal strength on the spectrum.From these, two or more fragments are selected in descending order ofstrength, and the average of the strength is defined as the expectedsensitivity of the MRR transitions. When a plurality of peptides isdetected from one protein, at least two peptides with the highestsensitivity are selected as standard peptides using the expectedsensitivity as an index.

Step (D) (Quantification of the Target Protein in the Test Sample). Step(D) comprises identifying, in the MRM chromatogram detected in step (C),a peptide derived from the target protein (a target biomarker ofinterest) that shows the same retention time as a peptide derived fromthe internal standard (an internal standard peptide), and quantifyingthe target protein in the test sample by comparing the peak area of theinternal standard peptide with the peak area of the target peptide. Thetarget protein can be quantified by utilizing a calibration curve of thestandard protein prepared beforehand.

The calibration curve can be prepared by the following method. First, arecombinant protein consisting of an amino acid sequence that isidentical to that of the target biomarker protein is digested with aprotease such as trypsin, as described above. Subsequently,precursor-fragment transition selection standards (PFTS) of a knownconcentration are individually labeled with two different types ofstable isotopes (i.e., one is labeled with a stable isomer used to labelan internal standard peptide (labeled with IS), whereas the other islabeled with a stable isomer used to label a target peptide (labeledwith T). A plurality of samples are produced by blending a certainamount of the IS-labeled PTFS with various concentrations of theT-labeled PTFS. These samples are placed in the aforementioned LC-MS/MSdevice to perform MRM analysis. The area ratio of the T-labeled PTFS tothe IS-labeled PTFS (T-labeled PTFS/IS-labeled PTFS) on the obtained MRMchromatogram is plotted against the amount of the T-labeled PTFS toprepare a calibration curve. The absolute amount of the target proteincontained in the test sample can be calculated by reference to thecalibration curve.

3. Antibodies and Labels (e.g., Fluorescent Moieties, Dyes)

In some embodiments, the invention provides methods and compositionsthat include labels for the highly sensitive detection and quantitationof the biomolecules of the invention, e.g., filamin A alone or incombination with at least one other prostate cancer related markerselected from the group consisting of filamin B, LY9, keratin 4, keratin7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSA,PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1. One skilled in theart will recognize that many strategies can be used for labeling targetmolecules to enable their detection or discrimination in a mixture ofparticles (e.g., labeled anti-filamin A antibody or labeled secondaryantibody, or labeled oligonucleotide probe that specifically hybridizesto filamin A mRNA). The labels may be attached by any known means,including methods that utilize non-specific or specific interactions oflabel and target. Labels may provide a detectable signal or affect themobility of the particle in an electric field. In addition, labeling canbe accomplished directly or through binding partners.

In some embodiments, the label comprises a binding partner that binds tothe biomarker of interest, where the binding partner is attached to afluorescent moiety. The compositions and methods of the invention mayutilize highly fluorescent moieties, e.g., a moiety capable of emittingat least about 200 photons when simulated by a laser emitting light atthe excitation wavelength of the moiety, wherein the laser is focused ona spot not less than about 5 microns in diameter that contains themoiety, and wherein the total energy directed at the spot by the laseris no more than about 3 microJoules. Moieties suitable for thecompositions and methods of the invention are described in more detailbelow.

In some embodiments, the invention provides a label for detecting abiological molecule comprising a binding partner for the biologicalmolecule that is attached to a fluorescent moiety, wherein thefluorescent moiety is capable of emitting at least about 200 photonswhen simulated by a laser emitting light at the excitation wavelength ofthe moiety, wherein the laser is focused on a spot not less than about 5microns in diameter that contains the moiety, and wherein the totalenergy directed at the spot by the laser is no more than about 3microJoules. In some embodiments, the moiety comprises a plurality offluorescent entities, e.g., about 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to8, 2 to 9, 2 to 10, or about 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, or3 to 10 fluorescent entities. In some embodiments, the moiety comprisesabout 2 to 4 fluorescent entities. In some embodiments, the biologicalmolecule is a protein or a small molecule. In some embodiments, thebiological molecule is a protein. The fluorescent entities can befluorescent dye molecules. In some embodiments, the fluorescent dyemolecules comprise at least one substituted indolium ring system inwhich the substituent on the 3-carbon of the indolium ring contains achemically reactive group or a conjugated substance. In someembodiments, the dye molecules are Alexa Fluor molecules selected fromthe group consisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor647, Alexa Fluor 680 or Alexa Fluor 700. In some embodiments, the dyemolecules are Alexa Fluor molecules selected from the group consistingof Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 680 or Alexa Fluor 700.In some embodiments, the dye molecules are Alexa Fluor 647 dyemolecules. In some embodiments, the dye molecules comprise a first typeand a second type of dye molecules, e.g., two different Alexa Fluormolecules, e.g., where the first type and second type of dye moleculeshave different emission spectra. The ratio of the number of first typeto second type of dye molecule can be, e.g., 4 to 1, 3 to 1, 2 to 1, 1to 1, 1 to 2, 1 to 3 or 1 to 4. The binding partner can be, e.g., anantibody.

In some embodiments, the invention provides a label for the detection ofa biological marker of the invention, wherein the label comprises abinding partner for the marker and a fluorescent moiety, wherein thefluorescent moiety is capable of emitting at least about 200 photonswhen simulated by a laser emitting light at the excitation wavelength ofthe moiety, wherein the laser is focused on a spot not less than about 5microns in diameter that contains the moiety, and wherein the totalenergy directed at the spot by the laser is no more than about 3microJoules. In some embodiments, the fluorescent moiety comprises afluorescent molecule. In some embodiments, the fluorescent moietycomprises a plurality of fluorescent molecules, e.g., about 2 to 10, 2to 8, 2 to 6, 2 to 4, 3 to 10, 3 to 8, or 3 to 6 fluorescent molecules.In some embodiments, the label comprises about 2 to 4 fluorescentmolecules. In some embodiments, the fluorescent dye molecules compriseat least one substituted indolium ring system in which the substituenton the 3-carbon of the indolium ring contains a chemically reactivegroup or a conjugated substance. In some embodiments, the fluorescentmolecules are selected from the group consisting of Alexa Fluor 488,Alexa Fluor 532, Alexa Fluor 647, Alexa Fluor 680 or Alexa Fluor 700. Insome embodiments, the fluorescent molecules are selected from the groupconsisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 680 or AlexaFluor 700. In some embodiments, the fluorescent molecules are AlexaFluor 647 molecules. In some embodiments, the binding partner comprisesan antibody. In some embodiments, the antibody is a monoclonal antibody.In other embodiments, the antibody is a polyclonal antibody.

In various embodiments, the binding partner for detecting a biomarker ofinterest, e.g., filamin A or filamin B, LY9, keratin 4, keratin 7,keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3 and PSA,is an antibody or antigen-binding fragment thereof. The term “antibody,”as used herein, is a broad term and is used in its ordinary sense,including, without limitation, to refer to naturally occurringantibodies as well as non-naturally occurring antibodies, including, forexample, single chain antibodies, chimeric, bifunctional and humanizedantibodies, as well as antigen-binding fragments thereof. An“antigen-binding fragment” of an antibody refers to the part of theantibody that participates in antigen binding. The antigen binding siteis formed by amino acid residues of the N-terminal variable (“V”)regions of the heavy (“H”) and light (“L”) chains. It will beappreciated that the choice of epitope or region of the molecule towhich the antibody is raised will determine its specificity, e.g., forvarious forms of the molecule, if present, or for total (e.g., all, orsubstantially all of the molecule).

Methods for producing antibodies are well-established. One skilled inthe art will recognize that many procedures are available for theproduction of antibodies, for example, as described in Antibodies, ALaboratory Manual, Ed Harlow and David Lane, Cold Spring HarborLaboratory (1988), Cold Spring Harbor, N.Y. One skilled in the art willalso appreciate that binding fragments or Fab fragments which mimicantibodies can also be prepared from genetic information by variousprocedures (Antibody Engineering: A Practical Approach (Borrebaeck, C.,ed.), 1995, Oxford University Press, Oxford; J. Immunol. 149, 3914-3920(1992)). Monoclonal and polyclonal antibodies to molecules, e.g.,proteins, and markers also commercially available (R and D Systems,Minneapolis, Minn.; HyTest, HyTest Ltd., Turku Finland; Abcam Inc.,Cambridge, Mass., USA, Life Diagnostics, Inc., West Chester, Pa., USA;Fitzgerald Industries International, Inc., Concord, Mass. 01742-3049USA; BiosPacific, Emeryville, Calif.).

In some embodiments, the antibody is a polyclonal antibody. In otherembodiments, the antibody is a monoclonal antibody.

Antibodies may be prepared by any of a variety of techniques known tothose of ordinary skill in the art (see, for example, Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).In general, antibodies can be produced by cell culture techniques,including the generation of monoclonal antibodies as described herein,or via transfection of antibody genes into suitable bacterial ormammalian cell hosts, in order to allow for the production ofrecombinant antibodies.

Monoclonal antibodies may be prepared using hybridoma methods, such asthe technique of Kohler and Milstein (Eur. J. Immunol. 6:511-519, 1976),and improvements thereto. These methods involve the preparation ofimmortal cell lines capable of producing antibodies having the desiredspecificity. Monoclonal antibodies may also be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. DNAencoding antibodies employed in the disclosed methods may be isolatedand sequenced using conventional procedures. Recombinant antibodies,antibody fragments, and/or fusions thereof, can be expressed in vitro orin prokaryotic cells (e.g. bacteria) or eukaryotic cells (e.g. yeast,insect or mammalian cells) and further purified as necessary using wellknown methods.

More particularly, monoclonal antibodies (MAbs) may be readily preparedthrough use of well-known techniques, such as those exemplified in U.S.Pat. No. 4,196,265, incorporated herein by reference. Typically, thistechnique involves immunizing a suitable animal with a selectedimmunogen composition, e.g., a purified or partially purified expressedprotein, polypeptide or peptide. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells. The methods for generating monoclonal antibodies (MAbs) generallybegin along the same lines as those for preparing polyclonal antibodies.Rodents such as mice and rats are preferred animals, however, the use ofrabbit, sheep or frog cells is also possible. The use of rats mayprovide certain advantages (Goding, 1986, pp. 60-61), but mice arepreferred, with the BALB/c mouse being most preferred as this is mostroutinely used and generally gives a higher percentage of stablefusions.

The animals are injected with antigen as described above. The antigenmay be coupled to carrier molecules such as keyhole limpet hemocyanin ifnecessary. The antigen would typically be mixed with adjuvant, such asFreund's complete or incomplete adjuvant. Booster injections with thesame antigen would occur at approximately two-week intervals. Followingimmunization, somatic cells with the potential for producing antibodies,specifically B lymphocytes (B cells), are selected for use in the MAbgenerating protocol. These cells may be obtained from biopsied spleens,tonsils or lymph nodes, or from a peripheral blood sample. Spleen cellsand peripheral blood cells are preferred, the former because they are arich source of antibody-producing cells that are in the dividingplasmablast stage, and the latter because peripheral blood is easilyaccessible. Often, a panel of animals will have been immunized and thespleen of the animal with the highest antibody titer will be removed andthe spleen lymphocytes obtained by homogenizing the spleen with asyringe.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones may then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. A sample of the hybridoma may beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,may then be tapped to provide MAbs in high concentration. The individualcell lines also may be cultured in vitro, where the MAbs are naturallysecreted into the culture medium from which they may be readily obtainedin high concentrations. MAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

Large amounts of the monoclonal antibodies of the present invention alsomay be obtained by multiplying hybridoma cells in vivo. Cell clones areinjected into mammals which are histocompatible with the parent cells,e.g., syngeneic mice, to cause growth of antibody-producing tumors.Optionally, the animals are primed with a hydrocarbon, especially oilssuch as pristane (tetramethylpentadecane) prior to injection.

In accordance with the present invention, fragments of the monoclonalantibody of the invention may be obtained from the monoclonal antibodyproduced as described above, by methods which include digestion withenzymes such as pepsin or papain and/or cleavage of disulfide bonds bychemical reduction. Alternatively, monoclonal antibody fragmentsencompassed by the present invention may be synthesized using anautomated peptide synthesizer.

Antibodies may also be derived from a recombinant antibody library thatis based on amino acid sequences that have been designed in silico andencoded by polynucleotides that are synthetically generated. Methods fordesigning and obtaining in silico-created sequences are known in the art(Knappik et al., J. Mol. Biol. 296:254:57-86, 2000; Krebs et al., J.Immunol. Methods 254:67-84, 2001; U.S. Pat. No. 6,300,064).

Digestion of antibodies to produce antigen-binding fragments thereof canbe performed using techniques well known in the art. For example, theproteolytic enzyme papain preferentially cleaves IgG molecules to yieldseveral fragments, two of which (the “F(ab)” fragments) each comprise acovalent heterodimer that includes an intact antigen-binding site. Theenzyme pepsin is able to cleave IgG molecules to provide severalfragments, including the “F(ab′).sub.2” fragment, which comprises bothantigen-binding sites. “Fv” fragments can be produced by preferentialproteolytic cleavage of an IgM, IgG or IgA immunoglobulin molecule, butare more commonly derived using recombinant techniques known in the art.The Fv fragment includes a non-covalent V.sub.H::V.sub.L heterodimerincluding an antigen-binding site which retains much of the antigenrecognition and binding capabilities of the native antibody molecule(Inbar et al., Proc. Natl. Acad. Sci. USA 69:2659-2662 (1972); Hochmanet al., Biochem. 15:2706-2710 (1976); and Ehrlich et al., Biochem.19:4091-4096 (1980)).

Antibody fragments that specifically bind to the polypeptide biomarkersdisclosed herein can also be isolated from a library of scFvs usingknown techniques, such as those described in U.S. Pat. No. 5,885,793.

A wide variety of expression systems are available in the art for theproduction of antibody fragments, including Fab fragments, scFv, VL andVHs. For example, expression systems of both prokaryotic and eukaryoticorigin may be used for the large-scale production of antibody fragments.Particularly advantageous are expression systems that permit thesecretion of large amounts of antibody fragments into the culturemedium. Eukaryotic expression systems for large-scale production ofantibody fragments and antibody fusion proteins have been described thatare based on mammalian cells, insect cells, plants, transgenic animals,and lower eukaryotes. For example, the cost-effective, large-scaleproduction of antibody fragments can be achieved in yeast fermentationsystems. Large-scale fermentation of these organisms is well known inthe art and is currently used for bulk production of several recombinantproteins.

Antibodies that bind to the polypeptide biomarkers employed in thepresent methods are well known to those of skill in the art and in somecases are available commercially or can be obtained without undueexperimentation.

In still other embodiments, particularly where oligonucleotides are usedas binding partners to detect and hybridize to mRNA biomarkers or othernucleic acid based biomarkers, the binding partners (e.g.,oligonucleotides) can comprise a label, e.g., a fluorescent moiety ordye. In addition, any binding partner of the invention, e.g., anantibody, can also be labeled with a fluorescent moiety. Thefluorescence of the moiety will be sufficient to allow detection in asingle molecule detector, such as the single molecule detectorsdescribed herein. A “fluorescent moiety,” as that term is used herein,includes one or more fluorescent entities whose total fluorescence issuch that the moiety may be detected in the single molecule detectorsdescribed herein. Thus, a fluorescent moiety may comprise a singleentity (e.g., a Quantum Dot or fluorescent molecule) or a plurality ofentities (e.g., a plurality of fluorescent molecules). It will beappreciated that when “moiety,” as that term is used herein, refers to agroup of fluorescent entities, e.g., a plurality of fluorescent dyemolecules, each individual entity may be attached to the binding partnerseparately or the entities may be attached together, as long as theentities as a group provide sufficient fluorescence to be detected.

Typically, the fluorescence of the moiety involves a combination ofquantum efficiency and lack of photobleaching sufficient that the moietyis detectable above background levels in a single molecule detector,with the consistency necessary for the desired limit of detection,accuracy, and precision of the assay. For example, in some embodiments,the fluorescence of the fluorescent moiety is such that it allowsdetection and/or quantitation of a molecule, e.g., a marker, at a limitof detection of less than about 10, 5, 4, 3, 2, 1, 0.1, 0.01, 0.001,0.00001, or 0.000001 pg/ml and with a coefficient of variation of lessthan about 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% orless, e.g., about 10% or less, in the instruments described herein. Insome embodiments, the fluorescence of the fluorescent moiety is suchthat it allows detection and/or quantitation of a molecule, e.g., amarker, at a limit of detection of less than about 5, 1, 0.5, 0.1, 0.05,0.01, 0.005, 0.001 pg/ml and with a coefficient of variation of lessthan about 10%, in the instruments described herein. “Limit ofdetection,” or LoD, as those terms are used herein, includes the lowestconcentration at which one can identify a sample as containing amolecule of the substance of interest, e.g., the first non-zero value.It can be defined by the variability of zeros and the slope of thestandard curve. For example, the limit of detection of an assay may bedetermined by running a standard curve, determining the standard curvezero value, and adding 2 standard deviations to that value. Aconcentration of the substance of interest that produces a signal equalto this value is the “lower limit of detection” concentration.

Furthermore, the moiety has properties that are consistent with its usein the assay of choice. In some embodiments, the assay is animmunoassay, where the fluorescent moiety is attached to an antibody;the moiety must have properties such that it does not aggregate withother antibodies or proteins, or experiences no more aggregation than isconsistent with the required accuracy and precision of the assay. Insome embodiments, fluorescent moieties that are preferred arefluorescent moieties, e.g., dye molecules that have a combination of 1)high absorption coefficient; 2) high quantum yield; 3) highphotostability (low photobleaching); and 4) compatibility with labelingthe molecule of interest (e.g., protein) so that it may be analyzedusing the analyzers and systems of the invention (e.g., does not causeprecipitation of the protein of interest, or precipitation of a proteinto which the moiety has been attached).

Any suitable fluorescent moiety may be used. Examples include, but arenot limited to, Alexa Fluor dyes (Molecular Probes, Eugene, Oreg.). TheAlexa Fluor dyes are disclosed in U.S. Pat. Nos. 6,977,305; 6,974,874;6,130,101; and 6,974,305 which are herein incorporated by reference intheir entirety. Some embodiments of the invention utilize a dye chosenfrom the group consisting of Alexa Fluor 647, Alexa Fluor 488, AlexaFluor 532, Alexa Fluor 555, Alexa Fluor 610, Alexa Fluor 680, AlexaFluor 700, and Alexa Fluor 750. Some embodiments of the inventionutilize a dye chosen from the group consisting of Alexa Fluor 488, AlexaFluor 532, Alexa Fluor 647, Alexa Fluor 700 and Alexa Fluor 750. Someembodiments of the invention utilize a dye chosen from the groupconsisting of Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 555, AlexaFluor 610, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750. Someembodiments of the invention utilize the Alexa Fluor 647 molecule, whichhas an absorption maximum between about 650 and 660 nm and an emissionmaximum between about 660 and 670 nm. The Alexa Fluor 647 dye is usedalone or in combination with other Alexa Fluor dyes.

In some embodiments, the fluorescent label moiety that is used to detecta biomarker in a sample using the analyzer systems of the invention is aquantum dot. Quantum dots (QDs), also known as semiconductornanocrystals or artificial atoms, are semiconductor crystals thatcontain anywhere between 100 to 1,000 electrons and range from 2-10 nm.Some QDs can be between 10-20 nm in diameter. QDs have high quantumyields, which makes them particularly useful for optical applications.QDs are fluorophores that fluoresce by forming excitons, which aresimilar to the excited state of traditional fluorophores, but have muchlonger lifetimes of up to 200 nanoseconds. This property provides QDswith low photobleaching. The energy level of QDs can be controlled bychanging the size and shape of the QD, and the depth of the QDs'potential. One optical feature of small excitonic QDs is coloration,which is determined by the size of the dot. The larger the dot, theredder, or more towards the red end of the spectrum the fluorescence.The smaller the dot, the bluer or more towards the blue end it is. Thebandgap energy that determines the energy and hence the color of thefluoresced light is inversely proportional to the square of the size ofthe QD. Larger QDs have more energy levels which are more closelyspaced, thus allowing the QD to absorb photons containing less energy,i.e., those closer to the red end of the spectrum. Because the emissionfrequency of a dot is dependent on the bandgap, it is possible tocontrol the output wavelength of a dot with extreme precision. In someembodiments the protein that is detected with the single moleculeanalyzer system is labeled with a QD. In some embodiments, the singlemolecule analyzer is used to detect a protein labeled with one QD andusing a filter to allow for the detection of different proteins atdifferent wavelengths.

F. Isolated Biomarkers

1. Isolated Polypeptide Biomarkers

One aspect of the invention pertains to isolated marker proteins andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise antibodies directed against amarker protein or a fragment thereof. In one embodiment, the nativemarker protein can be isolated from cells or tissue sources by anappropriate purification scheme using standard protein purificationtechniques. In another embodiment, a protein or peptide comprising thewhole or a segment of the marker protein is produced by recombinant DNAtechniques. Alternative to recombinant expression, such protein orpeptide can be synthesized chemically using standard peptide synthesistechniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theprotein is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized. The language “substantiallyfree of cellular material” includes preparations of protein in which theprotein is separated from cellular components of the cells from which itis isolated or recombinantly produced. Thus, protein that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10%, or 5% (by dry weight) ofheterologous protein (also referred to herein as a “contaminatingprotein”). When the protein or biologically active portion thereof isrecombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,10%, or 5% of the volume of the protein preparation. When the protein isproduced by chemical synthesis, it is preferably substantially free ofchemical precursors or other chemicals, i.e., it is separated fromchemical precursors or other chemicals which are involved in thesynthesis of the protein. Accordingly such preparations of the proteinhave less than about 30%, 20%, 10%, 5% (by dry weight) of chemicalprecursors or compounds other than the polypeptide of interest.

Biologically active portions of a marker protein include polypeptidescomprising amino acid sequences sufficiently identical to or derivedfrom the amino acid sequence of the marker protein, which include feweramino acids than the full length protein, and exhibit at least oneactivity of the corresponding full-length protein. Typically,biologically active portions comprise a domain or motif with at leastone activity of the corresponding full-length protein. A biologicallyactive portion of a marker protein of the invention can be a polypeptidewhich is, for example, 10, 25, 50, 100 or more amino acids in length.Moreover, other biologically active portions, in which other regions ofthe marker protein are deleted, can be prepared by recombinanttechniques and evaluated for one or more of the functional activities ofthe native form of the marker protein.

Preferred marker proteins are encoded by nucleotide sequences providedin the sequence listing. Other useful proteins are substantiallyidentical (e.g., at least about 40%, preferably 50%, 60%, 70%, 80%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) to one of these sequencesand retain the functional activity of the correspondingnaturally-occurring marker protein yet differ in amino acid sequence dueto natural allelic variation or mutagenesis.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in the sequence of a first amino acid ornucleic acid sequence for optimal alignment with a second amino ornucleic acid sequence). The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position.Preferably, the percent identity between the two sequences is calculatedusing a global alignment. Alternatively, the percent identity betweenthe two sequences is calculated using a local alignment. The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment the two sequences are the samelength. In another embodiment, the two sequences are not the samelength.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the BLASTN and BLASTX programs of Altschul, et al.(1990) J. Mol. Biol. 215:403-410. BLAST nucleotide searches can beperformed with the BLASTN program, score=100, wordlength=12 to obtainnucleotide sequences homologous to a nucleic acid molecules of theinvention. BLAST protein searches can be performed with the BLASTPprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to a protein molecules of the invention. To obtain gappedalignments for comparison purposes, a newer version of the BLASTalgorithm called Gapped BLAST can be utilized as described in Altschulet al. (1997) Nucleic Acids Res. 25:3389-3402, which is able to performgapped local alignments for the programs BLASTN, BLASTP and BLASTX.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules. When utilizing BLAST,Gapped BLAST, and PSI-Blast programs, the default parameters of therespective programs (e.g., BLASTX and BLASTN) can be used. See the NCBIwebsite. Another preferred, non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller, (1988) CABIOS 4:11-17. Such an algorithm isincorporated into the ALIGN program (version 2.0) which is part of theGCG sequence alignment software package. When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.Yet another useful algorithm for identifying regions of local sequencesimilarity and alignment is the FASTA algorithm as described in Pearsonand Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448. When usingthe FASTA algorithm for comparing nucleotide or amino acid sequences, aPAM120 weight residue table can, for example, be used with a k-tuplevalue of 2.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

Another aspect of the invention pertains to antibodies directed againsta protein of the invention. In preferred embodiments, the antibodiesspecifically bind a marker protein or a fragment thereof. The terms“antibody” and “antibodies” as used interchangeably herein refer toimmunoglobulin molecules as well as fragments and derivatives thereofthat comprise an immunologically active portion of an immunoglobulinmolecule, (i.e., such a portion contains an antigen binding site whichspecifically binds an antigen, such as a marker protein, e.g., anepitope of a marker protein). An antibody which specifically binds to aprotein of the invention is an antibody which binds the protein, butdoes not substantially bind other molecules in a sample, e.g., abiological sample, which naturally contains the protein. Examples of animmunologically active portion of an immunoglobulin molecule include,but are not limited to, single-chain antibodies (scAb), F(ab) andF(ab′)₂ fragments.

An isolated protein of the invention or a fragment thereof can be usedas an immunogen to generate antibodies. The full-length protein can beused or, alternatively, the invention provides antigenic peptidefragments for use as immunogens. The antigenic peptide of a protein ofthe invention comprises at least 8 (preferably 10, 15, 20, or 30 ormore) amino acid residues of the amino acid sequence of one of theproteins of the invention, and encompasses at least one epitope of theprotein such that an antibody raised against the peptide forms aspecific immune complex with the protein. Preferred epitopes encompassedby the antigenic peptide are regions that are located on the surface ofthe protein, e.g., hydrophilic regions. Hydrophobicity sequenceanalysis, hydrophilicity sequence analysis, or similar analyses can beused to identify hydrophilic regions. In preferred embodiments, anisolated marker protein or fragment thereof is used as an immunogen.

The invention provides polyclonal and monoclonal antibodies. The term“monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope. Preferred polyclonal and monoclonal antibodycompositions are ones that have been selected for antibodies directedagainst a protein of the invention. Particularly preferred polyclonaland monoclonal antibody preparations are ones that contain onlyantibodies directed against a marker protein or fragment thereof.Methods of making polyclonal, monoclonal, and recombinant antibody andantibody fragments are well known in the art.

2. Isolated Nucleic Acid Biomarkers

One aspect of the invention pertains to isolated nucleic acid molecules,including nucleic acids which encode a marker protein or a portionthereof. Isolated nucleic acids of the invention also include nucleicacid molecules sufficient for use as hybridization probes to identifymarker nucleic acid molecules, and fragments of marker nucleic acidmolecules, e.g., those suitable for use as PCR primers for theamplification of a specific product or mutation of marker nucleic acidmolecules. As used herein, the term “nucleic acid molecule” is intendedto include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules(e.g., mRNA) and analogs of the DNA or RNA generated using nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid molecule. In one embodiment, an “isolated” nucleic acidmolecule (preferably a protein-encoding sequences) is free of sequenceswhich naturally flank the nucleic acid (i.e., sequences located at the5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organismfrom which the nucleic acid is derived. For example, in variousembodiments, the isolated nucleic acid molecule can contain less thanabout 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotidesequences which naturally flank the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived. In anotherembodiment, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized. A nucleic acid molecule that is substantially free ofcellular material includes preparations having less than about 30%, 20%,10%, or 5% of heterologous nucleic acid (also referred to herein as a“contaminating nucleic acid”).

A nucleic acid molecule of the present invention can be isolated usingstandard molecular biology techniques and the sequence information inthe database records described herein. Using all or a portion of suchnucleic acid sequences, nucleic acid molecules of the invention can beisolated using standard hybridization and cloning techniques (e.g., asdescribed in Sambrook et al., ed., Molecular Cloning: A LaboratoryManual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989).

A nucleic acid molecule of the invention can be amplified using cDNA,mRNA, or genomic DNA as a template and appropriate oligonucleotideprimers according to standard PCR amplification techniques. The nucleicacid so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, nucleotidescorresponding to all or a portion of a nucleic acid molecule of theinvention can be prepared by standard synthetic techniques, e.g., usingan automated DNA synthesizer.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which has a nucleotidesequence complementary to the nucleotide sequence of a marker nucleicacid or to the nucleotide sequence of a nucleic acid encoding a markerprotein. A nucleic acid molecule which is complementary to a givennucleotide sequence is one which is sufficiently complementary to thegiven nucleotide sequence that it can hybridize to the given nucleotidesequence thereby forming a stable duplex.

Moreover, a nucleic acid molecule of the invention can comprise only aportion of a nucleic acid sequence, wherein the full length nucleic acidsequence comprises a marker nucleic acid or which encodes a markerprotein. Such nucleic acids can be used, for example, as a probe orprimer. The probe/primer typically is used as one or more substantiallypurified oligonucleotides. The oligonucleotide typically comprises aregion of nucleotide sequence that hybridizes under stringent conditionsto at least about 15, more preferably at least about 25, 50, 75, 100,125, 150, 175, 200, 250, 300, 350, or 400 or more consecutivenucleotides of a nucleic acid of the invention.

Probes based on the sequence of a nucleic acid molecule of the inventioncan be used to detect transcripts or genomic sequences corresponding toone or more markers of the invention. In certain embodiments, the probeshybridize to nucleic acid sequences that traverse splice junctions. Theprobe comprises a label group attached thereto, e.g., a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as part of a diagnostic test kit or panel for identifying cellsor tissues which express or mis-express the protein, such as bymeasuring levels of a nucleic acid molecule encoding the protein in asample of cells from a subject, e.g., detecting mRNA levels ordetermining whether a gene encoding the protein or its translationalcontrol sequences have been mutated or deleted.

The invention further encompasses nucleic acid molecules that differ,due to degeneracy of the genetic code, from the nucleotide sequence ofnucleic acids encoding a marker protein (e.g., protein having thesequence provided in the sequence listing), and thus encode the sameprotein.

It will be appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequence can existwithin a population (e.g., the human population). Such geneticpolymorphisms can exist among individuals within a population due tonatural allelic variation and changes known to occur in cancer. Anallele is one of a group of genes which occur alternatively at a givengenetic locus. In addition, it will be appreciated that DNApolymorphisms that affect RNA expression levels can also exist that mayaffect the overall expression level of that gene (e.g., by affectingregulation or degradation).

As used herein, the phrase “allelic variant” refers to a nucleotidesequence which occurs at a given locus or to a polypeptide encoded bythe nucleotide sequence.

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding a polypeptidecorresponding to a marker of the invention. Such natural allelicvariations can typically result in 1-5% variance in the nucleotidesequence of a given gene. Alternative alleles can be identified bysequencing the gene of interest in a number of different individuals.This can be readily carried out by using hybridization probes toidentify the same genetic locus in a variety of individuals. Any and allsuch nucleotide variations and resulting amino acid polymorphisms orvariations that are the result of natural allelic variation and that donot alter the functional activity are intended to be within the scope ofthe invention.

In another embodiment, an isolated nucleic acid molecule of theinvention is at least 15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250,300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1200, 1400, 1600,1800, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000, 4500, or morenucleotides in length and hybridizes under stringent conditions to amarker nucleic acid or to a nucleic acid encoding a marker protein. Asused herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 60% (65%, 70%, preferably 75%)identical to each other typically remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in sections 6.3.1-6.3.6 of Current Protocols in Molecular Biology,John Wiley & Sons, N.Y. (1989). A preferred, non-limiting example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65° C.

G. Biomarker Applications

The invention provides methods for diagnosing an abnormal prostatestate, e.g., BPH or an oncological disease state, e.g., prostate cancer,in a subject. The invention further provides methods for prognosing ormonitoring progression or monitoring response of an abnormal prostatestate, e.g., BPH or prostate cancer, to a therapeutic treatment duringactive treatment or watchful waiting.

In one aspect, the present invention constitutes an application ofdiagnostic information obtainable by the methods of the invention inconnection with analyzing, detecting, and/or measuring the prostatecancer biomarkers of the present invention, including filamin A alone orfilamin A in combination with one or more of filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1, which goes wellbeyond the discovered correlation between prostate cancer and thebiomarkers of the invention.

For example, when executing the methods of the invention for detectingand/or measuring a polypeptide biomarkers of the present invention, asdescribed herein, one contacts a biological sample with a detectionreagent, e.g, a monoclonal antibody, which selectively binds to thebiomarker of interest, forming a protein-protein complex, which is thenfurther detected either directly (if the antibody comprises a label) orindirectly (if a secondary detection reagent is used, e.g., a secondaryantibody, which in turn is labeled). Thus, the method of the inventiontransforms the polypeptide markers of the invention to a protein-proteincomplex that comprises either a detectable primary antibody or a primaryand further secondary antibody. Forming such protein-protein complexesis required in order to identify the presence of the biomarker ofinterest and necessarily changes the physical characteristics andproperties of the biomarker of interest as a result of conducting themethods of the invention.

The same principal applies when conducting the methods of the inventionfor detecting nucleic acid biomarkers of the invention. In particular,when amplification methods are used to detect a biomarker of theinvention (e.g., filamin A mRNA), the amplification process, in fact,results in the formation of a new population of amplicons—i.e.,molecules that are newly synthesized and which were not present in theoriginal biological sample, thereby physically transforming thebiological sample. Similarly, when hybridization probes are used todetect a target biomarker, a physical new species of molecules is ineffect created by the hybridization of the probes (optionally comprisinga label) to the target biomarker mRNA (or other nucleic acid), which isthen detected. Such polynucleotide products are effectively newlycreated or formed as a consequence of carrying out the method of theinvention.

The invention provides, in one embodiment, methods for diagnosing anoncological disorder, e.g., prostate cancer. The methods of the presentinvention can be practiced in conjunction with any other method used bythe skilled practitioner to prognose the occurrence or recurrence of anoncologic disorder and/or the survival of a subject being treated for anoncologic disorder. The diagnostic and prognostic methods providedherein can be used to determine if additional and/or more invasive testsor monitoring should be performed on a subject. It is understood that adisease as complex as an oncological disorder is rarely diagnosed usinga single test. Therefore, it is understood that the diagnostic,prognostic, and monitoring methods provided herein are typically used inconjunction with other methods known in the art. For example, themethods of the invention may be performed in conjunction with amorphological or cytological analysis of the sample obtained from thesubject, imaging analysis, and/or physical exam. Cytological methodswould include immunohistochemical or immunofluorescence detection (andquantitation if appropriate) of any other molecular marker either byitself, in conjunction with other markers. Other methods would includedetection of other markers by in situ PCR, or by extracting tissue andquantitating other markers by real time PCR. PCR is defined aspolymerase chain reaction.

Methods for assessing tumor progression during watchful waiting or theefficacy of a treatment regimen, e.g., chemotherapy, radiation therapy,surgery, hormone therapy, or any other therapeutic approach useful fortreating an oncologic disorder in a subject are also provided. In thesemethods the amount of marker in a pair of samples (a first sampleobtained from the subject at an earlier time point or prior to thetreatment regimen and a second sample obtained from the subject at alater time point, e.g., at a later time point when the subject hasundergone at least a portion of the treatment regimen) is assessed. Itis understood that the methods of the invention include obtaining andanalyzing more than two samples (e.g., 3, 4, 5, 6, 7, 8, 9, or moresamples) at regular or irregular intervals for assessment of markerlevels. Pairwise comparisons can be made between consecutive ornon-consecutive subject samples. Trends of marker levels and rates ofchange of marker levels can be analyzed for any two or more consecutiveor non-consecutive subject samples.

The invention also provides a method for determining whether anoncologic disorder, e.g., prostate cancer, is aggressive. The methodcomprises determining the amount of a marker present in a sample andcomparing the amount to a control amount of the marker present in one ormore control samples, as defined in Definitions, thereby determiningwhether an oncologic disorder is aggressive. Marker levels can becompared to marker levels in samples obtained at different times fromthe same subject or marker levels from normal or abnormal prostate statesubjects. A rapid increase in the level of marker may be indicative of amore aggressive cancer than a slow increase or no increase or change inthe marker level.

The methods of the invention may also be used to select a compound thatis capable of modulating, i.e., decreasing, the aggressiveness of anoncologic disorder, e.g., prostate cancer. In this method, a cancer cellis contacted with a test compound, and the ability of the test compoundto modulate the expression and/or activity of a marker in the inventionin the cancer cell is determined, thereby selecting a compound that iscapable of modulating aggressiveness of an oncologic disorder.

Using the methods described herein, a variety of molecules, may bescreened in order to identify molecules which modulate, e.g., increaseor decrease the expression and/or activity of a marker of the invention,e.g., filamin A alone or filamin A in combination with one or more ofPSA, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin19, tubulin-beta 3, filamin B (FLNB), and lymphocyte antigen 9 (LY9),PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1. Compounds soidentified can be provided to a subject in order to inhibit theaggressiveness of an oncologic disorder in the subject, to prevent therecurrence of an oncologic disorder in the subject, or to treat anoncologic disorder in the subject.

The present invention pertains to the field of predictive medicine inwhich diagnostic assays, prognostic assays, pharmacogenomics, andmonitoring clinical trials are used for prognostic (predictive) purposesto thereby treat an individual prophylactically. Accordingly, one aspectof the present invention relates to diagnostic assays for determiningthe level of expression of one or more marker proteins or nucleic acids,in order to determine whether an individual is at risk of developing adisease or disorder, such as, without limitation, an oncologicaldisorder, e.g., prostate cancer. Such assays can be used for prognosticor predictive purposes to thereby prophylactically treat an individualprior to the onset of the disorder.

Yet another aspect of the invention pertains to monitoring the influenceof agents (e.g., drugs or other therapeutic compounds) on the expressionor activity of a biomarker of the invention in clinical trials. Theseand other applications are described in further detail in the followingsections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence or change ofexpression level of a marker protein or nucleic acid in a biologicalsample involves obtaining a biological sample (e.g. an oncologicaldisorder-associated body fluid) from a test subject and contacting thebiological sample with a compound or an agent capable of detecting thepolypeptide or nucleic acid (e.g., mRNA, genomic DNA, or cDNA). Thedetection methods of the invention can thus be used to detect mRNA,protein, cDNA, or genomic DNA, for example, in a biological sample invitro as well as in vivo.

Methods provided herein for detecting the presence, absence, change ofexpression level of a marker protein or nucleic acid in a biologicalsample include obtaining a biological sample from a subject that may ormay not contain the marker protein or nucleic acid to be detected,contacting the sample with a marker-specific binding agent (i.e., one ormore marker-specific binding agents) that is capable of forming acomplex with the marker protein or nucleic acid to be detected, andcontacting the sample with a detection reagent for detection of themarker—marker-specific binding agent complex, if formed. It isunderstood that the methods provided herein for detecting an expressionlevel of a marker in a biological sample includes the steps to performthe assay. In certain embodiments of the detection methods, the level ofthe marker protein or nucleic acid in the sample is none or below thethreshold for detection.

The methods include formation of either a transient or stable complexbetween the marker and the marker-specific binding agent. The methodsrequire that the complex, if formed, be formed for sufficient time toallow a detection reagent to bind the complex and produce a detectablesignal (e.g., fluorescent signal, a signal from a product of anenzymatic reaction, e.g., a peroxidase reaction, a phosphatase reaction,a beta-galactosidase reaction, or a polymerase reaction).

In certain embodiments, all markers are detected using the same method.In certain embodiments, all markers are detected using the samebiological sample (e.g., same body fluid or tissue). In certainembodiments, different markers are detected using various methods. Incertain embodiments, markers are detected in different biologicalsamples.

2. Protein Detection

In certain embodiments of the invention, the marker to be detected is aprotein. Proteins are detected using a number of assays in which acomplex between the marker protein to be detected and the markerspecific binding agent would not occur naturally, for example, becauseone of the components is not a naturally occurring compound or themarker for detection and the marker specific binding agent are not fromthe same organism (e.g., human marker proteins detected usingmarker-specific binding antibodies from mouse, rat, or goat). In apreferred embodiment of the invention, the marker protein for detectionis a human marker protein. In certain detection assays, the humanmarkers for detection are bound by marker-specific, non-humanantibodies, thus, the complex would not be formed in nature. The complexof the marker protein can be detected directly, e.g., by use of alabeled marker-specific antibody that binds directly to the marker, orby binding a further component to the marker—marker-specific antibodycomplex. In certain embodiments, the further component is a secondmarker-specific antibody capable of binding the marker at the same timeas the first marker-specific antibody. In certain embodiments, thefurther component is a secondary antibody that binds to amarker-specific antibody, wherein the secondary antibody preferablylinked to a detectable label (e.g., fluorescent label, enzymatic label,biotin). When the secondary antibody is linked to an enzymaticdetectable label (e.g., a peroxidase, a phosphatase, abeta-galactosidase), the secondary antibody is detected by contactingthe enzymatic detectable label with an appropriate substrate to producea colorimetric, fluorescent, or other detectable, preferablyquantitatively detectable, product. Antibodies for use in the methods ofthe invention can be polyclonal, however, in a preferred embodimentmonoclonal antibodies are used. An intact antibody, or a fragment orderivative thereof (e.g., Fab or F(ab′)₂) can be used in the methods ofthe invention. Such strategies of marker protein detection are used, forexample, in ELISA, RIA, western blot, and immunofluorescence assaymethods.

In certain detection assays, the marker present in the biological samplefor detection is an enzyme and the detection reagent is an enzymesubstrate. For example, the enzyme can be a protease and the substratecan be any protein that includes an appropriate protease cleavage site.Alternatively, the enzyme can be a kinase and the substrate can be anysubstrate for the kinase. In preferred embodiments, the substrate whichforms a complex with the marker enzyme to be detected is not thesubstrate for the enzyme in a human subject.

In certain embodiments, the marker—marker-specific binding agent complexis attached to a solid support for detection of the marker. The complexcan be formed on the substrate or formed prior to capture on thesubstrate. For example, in an ELISA, RIA, immunoprecipitation assay,western blot, immunofluorescence assay, in gel enzymatic assay themarker for detection is attached to a solid support, either directly orindirectly. In an ELISA, RIA, or immunofluorescence assay, the marker istypically attached indirectly to a solid support through an antibody orbinding protein. In a western blot or immunofluorescence assay, themarker is typically attached directly to the solid support. For in-gelenzyme assays, the marker is resolved in a gel, typically an acrylamidegel, in which a substrate for the enzyme is integrated.

3. Nucleic Acid Detection

In certain embodiments of the invention, the marker is a nucleic acid.Nucleic acids are detected using a number of assays in which a complexbetween the marker nucleic acid to be detected and a marker-specificprobe would not occur naturally, for example, because one of thecomponents is not a naturally occurring compound. In certainembodiments, the analyte comprises a nucleic acid and the probecomprises one or more synthetic single stranded nucleic acid molecules,e.g., a DNA molecule, a DNA-RNA hybrid, a PNA, or a modified nucleicacid molecule containing one or more artificial bases, sugars, orbackbone moieties. In certain embodiments, the synthetic nucleic acid isa single stranded is a DNA molecule that includes a fluorescent label.In certain embodiments, the synthetic nucleic acid is a single strandedoligonucleotide molecule of about 12 to about 50 nucleotides in length.In certain embodiments, the nucleic acid to be detected is an mRNA andthe complex formed is an mRNA hybridized to a single stranded DNAmolecule that is complementary to the mRNA. In certain embodiments, anRNA is detected by generation of a DNA molecule (i.e., a cDNA molecule)first from the RNA template using the single stranded DNA thathybridizes to the RNA as a primer, e.g., a general poly-T primer totranscribe poly-A RNA. The cDNA can then be used as a template for anamplification reaction, e.g., PCR, primer extension assay, using amarker-specific probe. In certain embodiments, a labeled single strandedDNA can be hybridized to the RNA present in the sample for detection ofthe RNA by fluorescence in situ hybridization (FISH) or for detection ofthe RNA by northern blot.

For example, in vitro techniques for detection of mRNA include northernhybridizations, in situ hybridizations, and rtPCR. In vitro techniquesfor detection of genomic DNA include Southern hybridizations. Techniquesfor detection of mRNA include PCR, northern hybridizations and in situhybridizations. Methods include both qualitative and quantitativemethods.

A general principle of such diagnostic, prognostic, and monitoringassays involves preparing a sample or reaction mixture that may containa marker, and a probe, under appropriate conditions and for a timesufficient to allow the marker and probe to interact and bind, thusforming a complex that can be removed and/or detected in the reactionmixture. These assays can be conducted in a variety of ways known in theart, e.g., ELISA assay, PCR, FISH.

4. Detection of Expression Levels

Marker levels can be detected based on the absolute expression level ora normalized or relative expression level. Detection of absolute markerlevels may be preferable when monitoring the treatment of a subject orin determining if there is a change in the prostate cancer status of asubject. For example, the expression level of one or more markers can bemonitored in a subject undergoing treatment for prostate cancer, e.g.,at regular intervals, such a monthly intervals. A modulation in thelevel of one or more markers can be monitored over time to observetrends in changes in marker levels. Expression levels of the biomarkersof the invention, e.g., filamin A alone or in combination with one ormore of prostate specific antigen (PSA), filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1 in the subject maybe higher than the expression level of those markers in a normal sample,but may be lower than the prior expression level, thus indicating abenefit of the treatment regimen for the subject. Similarly, rates ofchange of marker levels can be important in a subject who is not subjectto active treatment for prostate cancer (e.g., watchful waiting).Changes, or not, in marker levels may be more relevant to treatmentdecisions for the subject than marker levels present in the population.Rapid changes in marker levels in a subject who otherwise appears tohave a normal prostate may be indicative of an abnormal prostate state,even if the markers are within normal ranges for the population.

As an alternative to making determinations based on the absoluteexpression level of the marker, determinations may be based on thenormalized expression level of the marker. Expression levels arenormalized by correcting the absolute expression level of a marker bycomparing its expression to the expression of a gene that is not amarker, e.g., a housekeeping gene that is constitutively expressed.Suitable genes for normalization include housekeeping genes such as theactin gene, or epithelial cell-specific genes. This normalization allowsthe comparison of the expression level in one sample, e.g., a patientsample, to another sample, e.g., a non-cancer sample, or between samplesfrom different sources.

Alternatively, the expression level can be provided as a relativeexpression level as compared to an appropriate control, e.g., populationcontrol, adjacent normal tissue control, earlier time point control,etc. Preferably, the samples used in the baseline determination will befrom non-cancer cells. The choice of the cell source is dependent on theuse of the relative expression level. Using expression found in normaltissues as a mean expression score aids in validating whether the markerassayed is cancer specific (versus normal cells). In addition, as moredata is accumulated, the mean expression value can be revised, providingimproved relative expression values based on accumulated data.Expression data from cancer cells provides a means for grading theseverity of the cancer state.

5. Diagnostic, Prognostic, and Treatment Methods

The invention provides methods for detecting an abnormal prostate statein a subject by

(1) contacting a biological sample from a subject with a panel of one ormore detection reagents wherein each detection reagent is specific forone prostate-cancer related protein; wherein the prostate-cancer relatedproteins are selected from the prostate-cancer related protein set asfollows: filamin A alone or filamin A in combination with one or more ofprostate specific antigen (PSA), filamin B, LY9, keratin 4, keratin 7,keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSM,PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1;

(2) measuring the amount of each prostate-cancer related marker detectedin the biological sample by each detection reagent; and

(3) comparing the level of expression of the one or more prostate-cancerrelated protein in the biological sample obtained from the subject witha level of expression of the one or more prostate-cancer related proteinin a control sample, thereby detecting an abnormal prostate state.

In certain embodiments, detecting an abnormal prostate state comprisesdiagnosing prostate cancer status in a subject. In certain embodiments,an abnormal prostate state comprises identifying a predisposition todeveloping prostate cancer.

The invention provides methods for monitoring the treatment of prostatecancer in a subject by

(1) contacting a first biological sample obtained from the subject priorto administering at least a portion of a treatment regimen to thesubject with a panel of one or more detection reagents wherein eachdetection reagent is specific for one prostate-cancer related protein;wherein the prostate-cancer related proteins are selected from theprostate protein set as follows: filamin A alone or in combination withone or more of prostate specific antigen (PSA), filamin B, LY9, keratin4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1;

(2) contacting a second biological sample obtained from the subjectafter administering at least a portion of a treatment regimen to thesubject with a panel of one or more detection reagents wherein eachdetection reagent is specific for one prostate-cancer related protein;wherein the prostate-cancer related proteins are selected from theprostate protein set as follows: filamin A alone or in combination withone or more of prostate specific antigen (PSA), filamin B, LY9, keratin4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1;

(3) measuring the amount of prostate-cancer related marker detected ineach the first biological sample and the second biological sample byeach detection reagent; and

(4) comparing the level of expression of the one or more prostate-cancerrelated markers in the first sample with the expression level of the oneor more prostate-cancer related markers in the second sample, therebymonitoring the treatment of prostate cancer in the subject.

The invention provides method of selecting for administration of activetreatment or against administration of active treatment of prostatecancer in a subject by

(1) contacting a first biological sample obtained from the subject priorto administering a treatment regimen to the subject with a panel of oneor more detection reagents wherein each detection reagent is specificfor one prostate-cancer related protein; wherein the prostate-cancerrelated proteins are selected from the prostate protein set as follows:filamin A alone or in combination with prostate specific antigen (PSA),filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, andPCGEM1;

(2) contacting a second biological sample obtained from the subjectprior to administering a treatment regimen to the subject with a panelof one or more detection reagents wherein each detection reagent isspecific for one prostate-cancer related protein; wherein theprostate-cancer related proteins are selected from the prostate proteinset as follows: filamin A alone or in combination with one or more ofprostate specific antigen (PSA), filamin B, LY9, keratin 4, keratin 7,keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSM,PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1;

(3) measuring the amount of prostate-cancer related marker detected ineach the first biological sample and the second biological sample byeach detection reagent; and

(4) comparing the level of expression of the one or more prostate-cancerrelated markers in the first sample with the expression level of the oneor more prostate-cancer related markers in the second sample, whereinselecting for administration of active treatment or againstadministration of active treatment of prostate cancer is based on thepresence or absence of changes in the level of expression of one or moremarkers between the first sample and the second sample.

In certain embodiments of the diagnostic and monitoring methods providedherein, one or more prostate-cancer related markers is two or moremarkers. In certain embodiments of the diagnostic and monitoring methodsprovided herein, one or more prostate-cancer related markers is three ormore markers. In certain embodiments of the diagnostic and monitoringmethods provided herein, one or more prostate-cancer related markers isfour or more markers. In certain embodiments of the diagnostic andmonitoring methods provided herein, one or more prostate-cancer relatedmarkers is five or more markers. In certain embodiments of thediagnostic and monitoring methods provided herein, one or moreprostate-cancer related markers is six or more markers. In certainembodiments of the diagnostic and monitoring methods provided herein,one or more prostate-cancer related markers is seven or more markers. Incertain embodiments of the diagnostic and monitoring methods providedherein, one or more prostate-cancer related markers is eight or moremarkers. In certain embodiments of the diagnostic and monitoring methodsprovided herein, one or more prostate-cancer related markers is nine ormore markers.

In certain embodiments of the diagnostic methods provided herein, anincrease in the level of expression of one or more prostate-cancerrelated markers selected from the group consisting of filamin A alone orin combination with one or more of prostate specific antigen (PSA),filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, andPCGEM1 in the biological sample as compared to the level of expressionof the one or more prostate-cancer related markers in a normal controlsample is an indication that the subject is afflicted with prostatecancer. In certain embodiments of the diagnostic methods providedherein, no increase in the detected expression level of filamin A or oneor more of prostate specific antigen (PSA), filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1 in the biologicalsample as compared to the expression level in a normal control sample isan indication that the subject is not afflicted with prostate cancer ornot predisposed to developing prostate cancer. In one embodiment, theage of the patient is also determined and used as a predictor variable.For example, increased patient age is an indication that the subject isafflicted with prostate cancer or is predisposed to developing prostatecancer.

In certain embodiments of the diagnostic methods provided herein, anincrease in the level of expression of one or more prostate-cancerrelated markers selected from the group consisting of filamin A alone orin combination with one or more of prostate specific antigen (PSA),filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, andPCGEM1 in the biological sample as compared to the level of expressionof the one or more prostate-cancer related markers in a normal controlsample is an indication that the subject is predisposed to developingprostate cancer. In one embodiment, the age of the patient is alsodetermined and used as a predictor variable. For example, increasedpatient age is an indication that the subject is afflicted with prostatecancer or is predisposed to developing prostate cancer.

In certain embodiments of the monitoring methods provided herein, noincrease in the detected level of expression of any of the one or moreprostate-cancer related markers selected from the group consisting offilamin A alone or in combination with one or more of prostate specificantigen (PSA), filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin15, keratin 18, keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF,HPG-1, PCA3, and PCGEM1 in the second sample as compared to the level ofexpression of the one or more prostate-cancer related markers in thefirst sample is an indication that the therapy is efficacious fortreating prostate cancer in the subject. In certain embodiments themonitoring methods provided herein, further comprise comparing the levelof expression of one or more prostate-cancer related markers selectedfrom the group consisting of filamin A alone or in combination with oneor more of prostate specific antigen (PSA), filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1 in the first sampleor the level of expression of one or more prostate-cancer relatedmarkers selected from the group consisting of filamin A alone or incombination with one or more of prostate specific antigen (PSA), filaminB, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1 inthe second sample with the expression of the one or more prostate-cancerrelated markers in a control sample.

In certain embodiments of the monitoring methods provided herein, anincrease in the level of expression of the one or more prostate-cancerrelated markers selected from the group consisting of filamin A alone orin combination with one or more of prostate specific antigen (PSA),filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, andPCGEM1 in the second sample as compared to the level of expression ofthe one or more prostate-cancer related markers in the first sample isan indication for selection of active treatment of prostate cancer inthe subject. In certain embodiments of the monitoring methods providedherein, no increase in the detected level of expression of any of theone or more prostate-cancer related markers selected from the groupconsisting of filamin A alone or in combination with one or more ofprostate specific antigen (PSA), filamin B, LY9, keratin 4, keratin 7,keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSM,PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1 in the second sample ascompared to the level of expression of the one or more prostate-cancerrelated markers in the first sample is an indication against selectionof active treatment of prostate cancer in the subject. In certainembodiments of the monitoring methods provided herein, wherein anincreased expression level of filamin A alone or in combination with oneor more of prostate specific antigen (PSA), filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1 in the secondsample as compared to the expression level in the first sample is anindication that the therapy is not efficacious in the treatment ofprostate cancer.

In certain embodiments of the diagnostic and monitoring methods providedherein, the one or more prostate-cancer related markers is selected fromthe group of filamin A alone or in combination with one or more ofprostate specific antigen (PSA), filamin B, LY9, keratin 4, keratin 7,keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSM,PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1. In certain embodiments ofthe diagnostic and monitoring methods provided herein, the one or moreprostate-cancer related markers comprise at least keratin 7, keratin 8,and keratin 15. In certain embodiments of the diagnostic and monitoringmethods provided herein, the one or more prostate-cancer related markersis selected from the group of keratin 7, keratin 15, and keratin 19. Incertain embodiments of the diagnostic and monitoring methods providedherein, the one or more prostate-cancer related markers comprise atleast keratin 7 or keratin 15. In certain embodiments of the diagnosticand monitoring methods provided herein, the one or more prostate-cancerrelated markers comprise at least keratin 4, keratin 7, keratin 8,keratin 15, keratin 18, and tubulin beta-3 in the biological sample iscompared to the level of the one or more prostate-cancer related markersin a normal control sample is indicative of a modulation in prostatecancer status.

In certain embodiments of the monitoring methods provided herein,modulation of the level of expression of the one or more prostate-cancerrelated markers selected from the group consisting of filamin A alone orin combination with one or more of prostate specific antigen (PSA),filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, andPCGEM1 in the second sample as compared to the level of expression ofthe corresponding marker(s) in the first sample is indicative of achange in prostate cancer status in response to treatment of theprostate cancer in the subject. In certain embodiments of the monitoringmethods provided herein, the methods further comprise comparing thelevel of expression of one or more prostate-cancer related markersselected from the group consisting of keratin 4, keratin 7, keratin 8,keratin 15, keratin 18, and tubulin beta-3 in the first sample; or thelevel of expression of one or more prostate-cancer related markersselected from the group consisting of keratin 4, keratin 7, keratin 8,keratin 15, keratin 18, and tubulin beta-3 in the second sample to thelevel of expression of one or more prostate-cancer related markers in anormal control sample.

In any of the aforementioned embodiments, the methods may also include astep of determining whether a subject having prostate cancer or who isbeing treated for prostate cancer is responsive to a particulartreatment. Such a step can include, for example, measuring the level ofexpression of one or more prostate-cancer related markers selected fromthe group consisting of filamin A alone or in combination with one ormore of prostate specific antigen (PSA), filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1 prior toadministering an anti-prostate cancer treatment, and measuring the levelof expression of one or more prostate-cancer related markers selectedfrom the group consisting of filamin A alone or in combination with oneor more of prostate specific antigen (PSA), filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1 after administeringthe anti-prostate cancer treatment, and comparing the expression levelbefore and after treatment. Determining that the prostate cancer isresponsive to the treatment if the expression level of the one or moremarkers is lower than before treatment as compared to after treatment.The method may further include the step of adjusting the treatment to ahigher dose in order to increase the responsiveness to the treatment, oradjusting the treatment to a lower dose in order to decrease theresponsiveness to the treatment.

In any of the aforementioned embodiments, the methods may also include astep of determining whether a subject having prostate cancer or who isbeing treated for prostate cancer is responsive to a particulartreatment. Such a step can include, for example, measuring the level ofexpression of one or more prostate-cancer related markers selected fromthe group consisting of filamin A alone or in combination with one ormore of prostate specific antigen (PSA), filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1 prior toadministering an anti-prostate cancer treatment, and measuring the levelof expression of one or more prostate-cancer related markers selectedfrom the group consisting of filamin A alone or in combination with oneor more of prostate specific antigen (PSA), filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1 after administeringthe anti-prostate cancer treatment, and comparing the expression levelbefore and after treatment. Determining that the prostate cancer isresponsive to the treatment if the expression level of the one or moremarkers is higher than before treatment as compared to after treatment.The method may further include the step of adjusting the treatment to ahigher dose in order to increase the responsiveness to the treatment, oradjusting the treatment to a lower dose in order to decrease theresponsiveness to the treatment.

In any of the aforementioned embodiments, the methods may also include astep of determining whether a subject having prostate cancer or who isbeing treated for prostate cancer is not responsive to a particulartreatment. Such a step can include, for example, measuring the level ofexpression of one or more prostate-cancer related markers selected fromthe group consisting of filamin A alone or in combination with one ormore of prostate specific antigen (PSA), filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1 prior toadministering an anti-prostate cancer treatment, and measuring the levelof expression of one or more prostate-cancer related markers selectedfrom the group consisting of filamin A alone or in combination with oneor more of prostate specific antigen (PSA), filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1 after administeringthe anti-prostate cancer treatment, and comparing the expression levelbefore and after treatment. Determining that the prostate cancer is notresponsive to the treatment if the expression level of the one or moremarkers is lower than before treatment as compared to after treatment.The method may further include the step of adjusting the treatment to ahigher dose in order to increase the responsiveness to the treatment.

In any of the aforementioned embodiments, the methods may also include astep of determining whether a subject having prostate cancer or who isbeing treated for prostate cancer is not responsive to a particulartreatment. Such a step can include, for example, measuring the level ofexpression of one or more prostate-cancer related markers selected fromthe group consisting of filamin A alone or in combination with one ormore of prostate specific antigen (PSA), filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1 prior toadministering an anti-prostate cancer treatment, and measuring the levelof expression of one or more prostate-cancer related markers selectedfrom the group consisting of filamin A alone or in combination with oneor more of prostate specific antigen (PSA), filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1 after administeringthe anti-prostate cancer treatment, and comparing the expression levelbefore and after treatment. Determining that the prostate cancer is notresponsive to the treatment if the expression level of the one or moremarkers is higher than before treatment as compared to after treatment.The method may further include the step of adjusting the treatment to ahigher dose in order to increase the responsiveness to the treatment.

In certain embodiments the diagnostic methods provided herein furthercomprise detecting the level of expression of prostate specific antigen(PSA) in the biological sample and preferably further comprise comparingthe level of expression of PSA in the biological sample to a PSAexpression level in a normal control sample. In certain embodiments, thecombination of PSA level with one or more of the prostate-cancer makerlevels increases the predictive value of the method.

In certain embodiments the monitoring methods provided herein furthercomprise detecting the level of expression of prostate specific antigen(PSA) in the first sample and the second sample, and preferably furthercomprising comparing the level of expression of PSA in the first samplewith the level of expression of PSA in the second sample. In certainmonitoring methods, the change in PSA level in combination with thechange in prostate-cancer maker level increases the predictive value ofthe method.

In certain embodiments the diagnostic and monitoring methods providedherein further comprise comparing the detected level of the one or moreprostate markers in the biological samples with one or more controlsamples wherein the control sample is one or more of a sample from thesame subject at an earlier time point than the biological sample, asample from a subject with benign prostatic hyperplasia (BPH), a samplefrom a subject with non-metastatic prostate cancer, a sample from asubject with metastatic prostate cancer, a sample from a subject withandrogen sensitive prostate cancer, a sample from a subject withandrogen insensitive prostate cancer, a sample from a subject withaggressive prostate cancer, and sample obtained from a subject withnon-aggressive prostate cancer. Comparison of the marker levels in thebiological samples with control samples from subjects with variousnormal and abnormal prostate states facilitates the differentiationbetween various prostate states including normal prostate and prostatecancer, benign prostate hyperplasia and prostate cancer, benign prostatehyperplasia and normal prostate, androgen dependent and androgenindependent prostate cancer, aggressive prostate cancer andnon-aggressive prostate cancer, aggressive prostate cancer andnon-aggressive prostate cancer, or between any two or more prostatestates including normal prostate, prostate cancer, benign prostatehyperplasia, androgen dependent prostate cancer, androgen independentprostate cancer, aggressive prostate cancer, non-aggressive prostatecancer, metastatic prostate cancer, and non-metastatic prostate cancer.

In certain embodiments the diagnostic and monitoring methods providedherein further comprising detecting the size of the prostate tumor inthe subject. In certain embodiments the monitoring methods providedherein further comprise detecting a change in the size or relativeaggressiveness of the tumor. In certain embodiments, the size of theprostate tumor in the subject is detected prior to administering the atleast a portion of a treatment regimen to the subject. In certainembodiments, the size of the prostate tumor in the subject is detectedafter administering the at least a portion of a treatment regimen to thesubject. Certain monitoring methods, further comprise comparing the sizeof the prostate tumor in the subject prior to administering the at leasta portion of a treatment regimen to the subject to the size of theprostate tumor in the subject after administering the at least a portionof a treatment regimen to the subject. Certain other embodiments of thediagnostic and monitoring methods further comprise determining theparticular stage or grade of prostate cancer, e.g., Gleason grade 1,grade 2, grade 3, grade 4, or grade 5 prostate cancer or TNMclassifications. In other embodiments, the present invention alsoinvolves the analysis and consideration of any clinical and/orpatient-related health data, for example, data obtained from anElectronic Medical Record (e.g., collection of electronic healthinformation about individual patients or populations relating to varioustypes of data, such as, demographics, medical history, medication andallergies, immunization status, laboratory test results, radiologyimages, vital signs, personal statistics like age and weight, andbilling information).

In certain embodiments the diagnostic and monitoring methods providedherein further comprising obtaining a subject sample.

In certain embodiments the diagnostic and monitoring methods providedherein further comprising selecting a treatment regimen for the subjectbased on the level of expression of one or more of the prostate-cancerrelated markers selected from the group consisting of filamin A alone orfilamin A in combination with one or more of filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1.

In certain embodiments the diagnostic and monitoring methods providedherein further comprise selecting a subject for having or beingsuspected of having prostate cancer.

In certain embodiments the diagnostic and monitoring methods providedherein further comprising treating the subject with a regimen includingone or more treatments selected from the group consisting of surgery,radiation, hormone therapy, antibody therapy, therapy with growthfactors, cytokines, and chemotherapy.

In certain embodiments the diagnostic and monitoring methods providedherein further comprise selecting the one or more specific treatmentregimens for the subject based on the results of the diagnostic andmonitoring methods provided herein. In one embodiment, a treatmentregimen known to be effective against prostate cancer having thebiomarker signature detected in the subject/sample is selected for thesubject. In certain embodiments, the treatment method is started,change, revised, or maintained based on the results from the diagnosticor prognostic methods of the invention, e.g., when it is determined thatthe subject is responding to the treatment regimen, or when it isdetermined that the subject is not responding to the treatment regimen,or when it is determined that the subject is insufficiently respondingto the treatment regimen. In certain embodiments, the treatment methodis changed based on the results from the diagnostic or prognosticmethods.

In certain other embodiments the diagnostic and monitoring methodsprovided herein further comprise introducing one or more specifictreatment regimens for the subject based on the results of thediagnostic and monitoring methods provided herein. In one embodiment, atreatment regimen known to be effective against prostate cancer isselected for the subject. In certain embodiments, the treatment methodis started, change, revised, or maintained based on the results from thediagnostic or prognostic methods of the invention, e.g., when it isdetermined that the subject is responding to the treatment regimen, orwhen it is determined that the subject is not responding to thetreatment regimen, or when it is determined that the subject isinsufficiently responding to the treatment regimen. In certainembodiments, the treatment method is changed based on the results fromthe diagnostic or prognostic methods.

In yet other embodiments the diagnostic and monitoring methods providedherein further comprise the step of administering a therapeuticallyeffective amount of an anti-prostate cancer therapy based on the resultsof the diagnostic and monitoring methods provided herein. In oneembodiment, a treatment regimen known to be effective against prostatecancer is selected for the subject. In certain embodiments, thetreatment method is administered based on the results from thediagnostic or prognostic methods of the invention, e.g., when it isdetermined that the subject expresses one or more biomarkers of theinvention (e.g., filamin A alone or filamin A in combination with one ormore of filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin 15,keratin 18, keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1,PCA3, and PCGEM1) above some threshold level that is indicative ofprostate cancer.

In yet other embodiments the diagnostic and monitoring methods providedherein further comprise the step of administering a therapeuticallyeffective amount of an anti-prostate cancer therapy based on the resultsof the diagnostic and monitoring methods provided herein. In oneembodiment, a treatment regimen known to be effective against prostatecancer is selected for the subject. In certain embodiments, thetreatment method is administered based on the results from thediagnostic or prognostic methods of the invention, e.g., when it isdetermined that the subject expresses one or more biomarkers of theinvention (e.g., filamin A alone or filamin A in combination with one ormore of filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin 15,keratin 18, keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1,PCA3, and PCGEM1) below some threshold level that is indicative ofprostate cancer.

In yet other embodiments the diagnostic and monitoring methods providedherein further comprise the step of increasing, decreasing, or changingthe dose of an anti-prostate cancer therapy based on the results of thediagnostic and monitoring methods provided herein. In one embodiment, atreatment regimen known to be effective against prostate cancer isselected for the subject. In certain embodiments, the treatment methodis administered based on the results from the diagnostic or prognosticmethods of the invention, e.g., when it is determined that the subjectexpresses one or more biomarkers of the invention (e.g., filamin A aloneor filamin A in combination with one or more of filamin B, LY9, keratin4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1) abovesome threshold level that is indicative of prostate cancer.

In yet other embodiments the diagnostic and monitoring methods providedherein further comprise the step of increasing, decreasing, or changingthe dose of an anti-prostate cancer therapy based on the results of thediagnostic and monitoring methods provided herein. In one embodiment, atreatment regimen known to be effective against prostate cancer isselected for the subject. In certain embodiments, the treatment methodis administered based on the results from the diagnostic or prognosticmethods of the invention, e.g., when it is determined that the subjectexpresses one or more biomarkers of the invention (e.g., filamin A aloneor filamin A in combination with one or more of filamin B, LY9, keratin4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1) belowsome threshold level that is indicative of prostate cancer.

In certain embodiments, a change in the treatment regimen compriseschanging a hormone based therapy treatment. In certain embodiments,treatments for prostate cancer include one or more of surgery,radiation, hormone therapy, antibody therapy, therapy with growthfactors, cytokines, or chemotherapy based on the results of a method ofany one of claims 1-64 for an interval prior to performing a subsequentdiagnostic, prognostic, or monitoring method provided herein.

In certain embodiments of the diagnostic and monitoring methods providedherein, the method further comprises isolating a component of thebiological sample.

In certain embodiments of the diagnostic and monitoring methods providedherein, the method further comprises labeling a component of thebiological sample.

In certain embodiments of the diagnostic and monitoring methods providedherein, the method further comprises amplifying a component of abiological sample.

In certain embodiments of the diagnostic and monitoring methods providedherein, the method comprises forming a complex with a probe and acomponent of a biological sample. In certain embodiments, forming acomplex with a probe comprises forming a complex with at least onenon-naturally occurring reagent. In certain embodiments of thediagnostic and monitoring methods provided herein, the method comprisesprocessing the biological sample. In certain embodiments of thediagnostic and monitoring methods provided herein, the method ofdetecting a level of at least two markers comprises a panel of markers.In certain embodiments of the diagnostic and monitoring methods providedherein, the method of detecting a level comprises attaching the markerto be detected to a solid surface.

The invention provides methods of selecting for administration of activetreatment or against administration of active treatment of prostatecancer in a subject comprising:

(1) detecting a level of one or more markers selected from the groupconsisting of filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin15, keratin 18, keratin 19, and tubulin-beta in a first sample obtainedfrom the subject having prostate cancer at a first time wherein thesubject has not been actively treated for prostate cancer;

(2) detecting a level of one or more markers selected from the groupconsisting of filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin15, keratin 18, keratin 19, and tubulin-beta 3 in a second sampleobtained from the subject at a second time, e.g., wherein the subjecthas not been actively treated;

(3) comparing the level of one or more markers selected from the groupconsisting of filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin15, keratin 18, keratin 19, and tubulin-beta 3 in the first sample withthe level of one or more markers selected from the group consisting offilamin B, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, and tubulin-beta 3 in the second sample;

wherein selecting for administration of active treatment or againstadministration of active treatment of prostate cancer is based on thepresence or absence of changes in the level of expression of one or moremarkers between the first sample and the second sample.

In certain embodiments, the method further comprising obtaining a thirdsample obtained from the subject at a third time (e.g., wherein thesubject has not been actively treated), detecting a level of one or moremarkers selected from the group consisting of filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, andtubulin-beta 3 in the third sample, and comparing the level of one ormore markers selected from the group consisting of filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, andtubulin-beta 3 in the third sample with the level of the one or moremarkers in the first sample and/or the one or more markers in the secondsample.

In certain embodiments, an increased level of filamin A alone or filaminA in combination with one or more of filamin B, LY9, keratin 4, keratin7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSM,PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1, in the second sample ascompared to the level of the markers in the first sample is anindication that the therapy is not efficacious in the treatment ofprostate cancer.

In certain embodiments, an increased level of filamin A alone or filaminA in combination with one or more of filamin B, LY9, keratin 4, keratin7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, PSM,PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1, in the second sample ascompared to the level of the markers in the first sample is anindication for selecting active treatment for prostate cancer.

In certain embodiments, the method further comprises comparing the levelof one or more markers selected from the group consisting of ilamin Aalone or filamin A in combination with one or more of filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1, inthe first sample or the level of one or more markers selected from thegroup consisting of ilamin A alone or filamin A in combination with oneor more of filamin B, LY9, keratin 4, keratin 7, keratin 8, keratin 15,keratin 18, keratin 19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1,PCA3, and PCGEM1, in the second sample with the level of one or more ofilamin A alone or filamin A in combination with one or more of filaminB, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1,in a control sample. In certain embodiments, the method comprisesdetecting the level of filamin A in combination with one or more ofkeratin 4, keratin 7, keratin 8, keratin 15, keratin 18, and tubulinbeta-3 in the first sample; detecting the level of filamin A incombination with one or more of keratin 4, keratin 7, keratin 8, keratin15, keratin 18, and tubulin beta-3 in the second sample; and comparingthe level of filamin A in combination with one or more of one or more ofkeratin 4, keratin 7, keratin 8, keratin 15, keratin 18, and tubulinbeta-3 in the second sample with the one or more of the level of filaminA in combination with keratin 4, keratin 7, keratin 8, keratin 15,keratin 18, and tubulin beta-3 in the first sample. In certainembodiments, the method comprises detection of a subset of keratins suchas keratin 7, keratin 8, and keratin 15; keratin 7, 15, and 19 incombination with filamin A; and keratin 7 or keratin 15. In certainembodiments, the method further comprises comparing the level of one ormore of keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, andtubulin beta-3 in combination with filamin A in the first sample; or thelevel of expression of one or more of keratin 4, keratin 7, keratin 8,keratin 15, keratin 18, and tubulin beta-3 in combination with filamin Ain the second sample to the level of one or more of keratin 4, keratin7, keratin 8, keratin 15, keratin 18, and tubulin beta-3 in combinationwith filamin A in a control sample.

In certain embodiments, no change in the level of expression of one ormore markers selected from the group consisting of filamin A alone orfilamin A in combination with one or more of filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1, between the firstsample and the second sample is an indication for selecting againstactive treatment for prostate cancer.

In certain embodiments, the methods further comprise detecting the levelof prostate specific antigen (PSA) in the first sample and the secondsample, and then preferably further comprising comparing the level ofPSA in the first sample with the level of PSA in the second sample.

In certain embodiments, a decrease in the level of one or more offilamin A alone or filamin A in combination with one or more of filaminB, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1,in the second sample as compared to the level of one or more of filaminA alone or filamin A in combination with one or more of filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1, inthe first sample in combination with a decrease in the level of PSA inthe second sample as compared to the level of PSA in the first samplehas greater predictive value that the therapy is efficacious in treatingprostate cancer in the subject than analysis of a single marker alone.

In certain embodiments, a decrease in the level of one or more offilamin A alone or filamin A in combination with one or more of filaminB, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin19, tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1,in the second sample as compared to the level of one or more of filaminA alone or filamin A in combination with one or more of filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1, inthe first sample in combination with a decrease in the level ofexpression of PSA in the second sample as compared to the level of PSAin the first sample has greater predictive value for selecting againstactive treatment for prostate cancer than analysis of a single markeralone.

6. Monitoring Clinical Trials

Monitoring the influence of agents (e.g., drug compounds) on the levelof expression of a marker of the invention can be applied not only inbasic drug screening or monitoring the treatment of a single subject,but also in clinical trials. For example, the effectiveness of an agentto affect marker expression can be monitored in clinical trials ofsubjects receiving treatment for an oncological disorder. In a preferredembodiment, the present invention provides a method for monitoring theeffectiveness of treatment of a subject with an agent (e.g., an agonist,antagonist, peptidomimetic, protein, peptide, nucleic acid, smallmolecule, or other drug candidate) comprising the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of one or moreselected markers of the invention (e.g., filamin B, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta3, optionally in combination with PSA) in the pre-administration sample;(iii) obtaining one or more post-administration samples from thesubject; (iv) detecting the level of expression of the marker(s) in thepost-administration samples; (v) comparing the level of expression ofthe marker(s) in the pre-administration sample with the level ofexpression of the marker(s) in the post-administration sample orsamples; and (vi) altering the administration of the agent to thesubject accordingly. For example, increased expression of the markergene(s) during the course of treatment may indicate ineffective dosageand the desirability of increasing the dosage. Conversely, decreasedexpression of the marker gene(s) may indicate efficacious treatment andno need to change dosage.

H. Treatment/Therapeutics

The present invention provides methods for use of one or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, or 9) markers selected from the group consisting offilamin A, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, tubulin-beta 3, filamin B (FLNB), and lymphocyte antigen 9(LY9) to treat disease states in a subject, e.g., a mammal, e.g., ahuman.

The present invention also provides methods for treatment of a subjectwith prostate cancer with a therapeutic, e.g., a nucleic acid basedtherapeutic, that modulates (e.g., reduces, or increases, and preferablyreduces) the expression or activity of one or more (e.g., 1, 2, 3, 4, 5,6, 7, 8, or 9) markers selected from the group consisting of filamin Aalone or filamin A in combination with one or more of filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, PSM, PSCA, TMPRSS2, PDEF, HPG-1, PCA3, and PCGEM1.

The invention also provides methods for selection and/or administrationof known treatment agents, especially hormone based therapies vs.non-hormone based therapies, and aggressive or active treatment vs.“watchful waiting”, depending on the detection of a change in the levelof one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9) markers selectedfrom the group consisting of filamin A, keratin 4, keratin 7, keratin 8,keratin 15, keratin 18, keratin 19, tubulin-beta 3, filamin B (FLNB),and lymphocyte antigen 9 (LY9), as compared to a control. The selectionof treatment regimens can further include the detection of PSA to assistin selection of the therapeutic methods. Selection of treatment methodscan also include other diagnostic considerations and patientcharacteristics including results from imaging studies, tumor size orgrowth rates, risk of poor outcomes, disruption of daily activities, andage, Gleason scores (e.g., grade 1, grade 2, grade 3, grade 4, or grade5 prostate cancer), TNM classifications, clinical and/or patient-relatedhealth data (e.g., data obtained from an Electronic Medical Record(e.g., collection of electronic health information about individualpatients or populations relating to various types of data, such as,demographics, medical history, medication and allergies, immunizationstatus, laboratory test results, radiology images, vital signs, personalstatistics like age and weight, and billing information).

As used herein, the term “aggressive oncological disorder”, such asaggressive prostate cancer, refers to an oncological disorder involvinga fast-growing tumor. An aggressive oncological disorder typically doesnot respond, responds poorly, or loses response to therapeutictreatment. For example, an prostate cancer may be considered to becomean aggressive prostate cancer upon loss of response to hormone therapy,necessitating treatment with chemotherapy, surgery, and/or radiation. Asused herein, an aggressive prostate cancer, for example, is one thatwill likely or has metastasized. As used herein, an aggressive prostatecancer is one that will result in significant changes in quality of lifeas the tumor grows. Active treatment is therapeutically indicated for anaggressive oncological disorder, e.g., aggressive prostate cancer.

As used herein, the term “non-aggressive oncological disorder” such as anon-aggressive prostate cancer, refers to an oncological disorderinvolving a slow-growing tumor. A non-aggressive oncological disordertypically responds favorably or moderately to therapeutic treatment orgrows so slowly that immediate treatment is not warranted. Anon-aggressive prostate tumor is one that a person skilled in the art,e.g., an oncologist, may decide to not actively treat with routineinterventions for the treatment of cancer, e.g., chemotherapy,radiation, surgery, as the active treatment may do more harm than thedisease, particularly in an older subject. A non-aggressive prostatetumor is one that a person skilled in the art may decide to monitor with“watchful waiting” rather than subjecting the person to any activetherapeutic interventions to alter the presence or growth of the tumor(e.g., radiation, surgery, chemotherapy, hormone therapy).

1. Nucleic Acid Therapeutics

Nucleic acid therapeutics are well known in the art. Nucleic acidtherapeutics include both single stranded and double stranded (i.e.,nucleic acid therapeutics having a complementary region of at least 15nucleotides in length that may be one or two nucleic acid strands)nucleic acids that are complementary to a target sequence in a cell.Nucleic acid therapeutics can be delivered to a cell in culture, e.g.,by adding the nucleic acid to culture media either alone or with anagent to promote uptake of the nucleic acid into the cell. Nucleic acidtherapeutics can be delivered to a cell in a subject, i.e., in vivo, byany route of administration. The specific formulation will depend on theroute of administration.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

Sequences can be “fully complementary” with respect to each when thereis base-pairing of the nucleotides of the first nucleotide sequence withthe nucleotides of the second nucleotide sequence over the entire lengthof the first and second nucleotide sequences. However, where a firstsequence is referred to as “substantially complementary” with respect toa second sequence herein, the two sequences can be fully complementary,or they may form one or more, but generally not more than 4, 3 or 2mismatched base pairs upon hybridization, while retaining the ability tohybridize under the conditions most relevant to their ultimateapplication. However, where two oligonucleotides are designed to form,upon hybridization, one or more single stranded overhangs as is commonin double stranded nucleic acid therapeutics, such overhangs shall notbe regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,may yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary”, and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenan antisense nucleic acid or the antisense strand of dsRNA and a targetsequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding filamin B, LY9, a keratin, tubulin-beta3, or PSA) including a 5′ UTR, an open reading frame (ORF), or a 3′ UTR.For example, a polynucleotide is complementary to at least a part offilamin B, LY9, a keratin, tubulin-beta 3, or PSA mRNA if the sequenceis substantially complementary to a non-interrupted portion of an mRNAencoding filamin B, LY9, a keratin, tubulin-beta 3, or PSA.

Nucleic acid therapeutics typically include chemical modifications toimprove their stability and to modulate their pharmacokinetic andpharmacodynamic properties. For example, the modifications on thenucleotides can include, but are not limited to, LNA, HNA, CeNA,2′-hydroxyl, and combinations thereof.

Nucleic acid therapeutics may further comprise at least onephosphorothioate or methylphosphonate internucleotide linkage. Thephosphorothioate or methylphosphonate internucleotide linkagemodification may occur on any nucleotide of the sense strand orantisense strand or both (in nucleic acid therapeutics including a sensestrand) in any position of the strand. For instance, the internucleotidelinkage modification may occur on every nucleotide on the sense strandor antisense strand; each internucleotide linkage modification may occurin an alternating pattern on the sense strand or antisense strand; orthe sense strand or antisense strand may contain both internucleotidelinkage modifications in an alternating pattern. The alternating patternof the internucleotide linkage modification on the sense strand may bethe same or different from the antisense strand, and the alternatingpattern of the internucleotide linkage modification on the sense strandmay have a shift relative to the alternating pattern of theinternucleotide linkage modification on the antisense strand.

A. Single Stranded Therapeutics

Antisense nucleic acid therapeutic agent single stranded nucleic acidtherapeutics, typically about 16 to 30 nucleotides in length and arecomplementary to a target nucleic acid sequence in the target cell,either in culture or in an organism.

Patents directed to antisense nucleic acids, chemical modifications, andtherapeutic uses are provided, for example, in U.S. Pat. No. 5,898,031related to chemically modified RNA-containing therapeutic compounds, andU.S. Pat. No. 6,107,094 related methods of using these compounds astherapeutic agent. U.S. Pat. No. 7,432,250 related to methods oftreating patients by administering single-stranded chemically modifiedRNA-like compounds; and U.S. Pat. No. 7,432,249 related topharmaceutical compositions containing single-stranded chemicallymodified RNA-like compounds. U.S. Pat. No. 7,629,321 is related tomethods of cleaving target mRNA using a single-stranded oligonucleotidehaving a plurality RNA nucleosides and at least one chemicalmodification. Each of the patents listed in the paragraph areincorporated herein by reference.

B. Double Stranded Therapeutics

In many embodiments, the duplex region is 15-30 nucleotide pairs inlength. In some embodiments, the duplex region is 17-23 nucleotide pairsin length, 17-25 nucleotide pairs in length, 23-27 nucleotide pairs inlength, 19-21 nucleotide pairs in length, or 21-23 nucleotide pairs inlength.

In certain embodiments, each strand has 15-30 nucleotides.

The RNAi agents that are used in the methods of the invention includeagents with chemical modifications as disclosed, for example, inPublications WO 2009/073809 and WO/2012/037254, the entire contents ofeach of which are incorporated herein by reference.

Nucleic acid therapeutic agents for use in the methods of the inventionalso include double stranded nucleic acid therapeutics. An “RNAi agent,”“double stranded RNAi agent,” double-stranded RNA (dsRNA) molecule, alsoreferred to as “dsRNA agent,” “dsRNA”, “siRNA”, “iRNA agent,” as usedinterchangeably herein, refers to a complex of ribonucleic acidmolecules, having a duplex structure comprising two anti-parallel andsubstantially complementary, as defined below, nucleic acid strands. Asused herein, an RNAi agent can also include dsiRNA (see, e.g., US Patentpublication 20070104688, incorporated herein by reference). In general,the majority of nucleotides of each strand are ribonucleotides, but asdescribed herein, each or both strands can also include one or morenon-ribonucleotides, e.g., a deoxyribonucleotide and/or a modifiednucleotide. In addition, as used in this specification, an “RNAi agent”may include ribonucleotides with chemical modifications; an RNAi agentmay include substantial modifications at multiple nucleotides. Suchmodifications may include all types of modifications disclosed herein orknown in the art. Any such modifications, as used in a siRNA typemolecule, are encompassed by “RNAi agent” for the purposes of thisspecification and claims. The RNAi agents that are used in the methodsof the invention include agents with chemical modifications asdisclosed, for example, in U.S. Provisional Application No. 61/561,710,filed on Nov. 18, 2011, International Application No. PCT/US2011/051597,filed on Sep. 15, 2010, and PCT Publication WO 2009/073809, the entirecontents of each of which are incorporated herein by reference. The twostrands forming the duplex structure may be different portions of onelarger RNA molecule, or they may be separate RNA molecules. Where thetwo strands are part of one larger molecule, and therefore are connectedby an uninterrupted chain of nucleotides between the 3′-end of onestrand and the 5′-end of the respective other strand forming the duplexstructure, the connecting RNA chain is referred to as a “hairpin loop.”Where the two strands are connected covalently by means other than anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′-end of the respective other strand forming the duplex structure,the connecting structure is referred to as a “linker.” The RNA strandsmay have the same or a different number of nucleotides. The maximumnumber of base pairs is the number of nucleotides in the shortest strandof the dsRNA minus any overhangs that are present in the duplex. Inaddition to the duplex structure, an RNAi agent may comprise one or morenucleotide overhangs. The term “siRNA” is also used herein to refer toan RNAi agent as described above.

In another aspect, the agent is a single-stranded antisense RNAmolecule. An antisense RNA molecule is complementary to a sequencewithin the target mRNA. Antisense RNA can inhibit translation in astoichiometric manner by base pairing to the mRNA and physicallyobstructing the translation machinery, see Dias, N. et al., (2002) MolCancer Ther 1:347-355. The antisense RNA molecule may have about 15-30nucleotides that are complementary to the target mRNA. For example, theantisense RNA molecule may have a sequence of at least 15, 16, 17, 18,19, 20 or more contiguous nucleotides complementary to the filamin B orLY9 sequences provided herein.

The term “antisense strand” refers to the strand of a double strandedRNAi agent which includes a region that is substantially complementaryto a target sequence (e.g., a human TTR mRNA). As used herein, the term“region complementary to part of an mRNA encoding transthyretin” refersto a region on the antisense strand that is substantially complementaryto part of a TTR mRNA sequence. Where the region of complementarity isnot fully complementary to the target sequence, the mismatches are mosttolerated in the terminal regions and, if present, are generally in aterminal region or regions, e.g., within 6, 5, 4, 3, or 2 nucleotides ofthe 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

The invention also includes molecular beacon nucleic acids having atleast one region which is complementary to a nucleic acid of theinvention, such that the molecular beacon is useful for quantitating thepresence of the nucleic acid of the invention in a sample. A “molecularbeacon” nucleic acid is a nucleic acid comprising a pair ofcomplementary regions and having a fluorophore and a fluorescentquencher associated therewith. The fluorophore and quencher areassociated with different portions of the nucleic acid in such anorientation that when the complementary regions are annealed with oneanother, fluorescence of the fluorophore is quenched by the quencher.When the complementary regions of the nucleic acid are not annealed withone another, fluorescence of the fluorophore is quenched to a lesserdegree. Molecular beacon nucleic acids are described, for example, inU.S. Pat. No. 5,876,930.

I. Drug Screening

As noted above, sets of biomarkers whose expression levels correlatewith one or more selected prostate disease characteristics (e.g.,prostate cancer progression) are attractive targets for identificationof new therapeutic agents via screens to detect compounds or entitiesthat inhibit or enhance expression of these biomarker genes and/or theirproducts. Accordingly, the present invention provides methods for theidentification of compounds potentially useful for modulating prostatecancer progression. In particular, the present invention providesmethods for the identification of compounds potentially useful formodulating prostate cancer progression wherein the compounds modulate(e.g., increase or decrease, preferably decrease or inhibit) theexpression of filamin A, and/or filamin A in combination with otherbiomarkers, including prostate specific antigen (PSA), filamin B, LY9,keratin 4, keratin 7, keratin 8, keratin 15, kertin 18, keratin 19, andtubulin-beta 3.

Such assays typically comprise a reaction between a marker of theinvention and one or more assay components. The other components may beeither the test compound itself, or a combination of test compounds anda natural binding partner of a marker of the invention. Compoundsidentified via assays such as those described herein may be useful, forexample, for modulating, e.g., inhibiting, ameliorating, treating, orpreventing the disease. Compounds identified for modulating theexpression level of one or more of keratin 4, keratin 7, keratin 8,keratin 15, keratin 18, keratin 19, tubulin-beta 3, filamin B, or LY9;optionally in combination with PSA, are preferably further tested foractivity useful in the treatment of cancer, preferably prostate cancer,e.g., inhibiting tumor cell growth, inhibiting tumor angiogenesis,inducing tumor cell apoptosis, etc.

The test compounds used in the screening assays of the present inventionmay be obtained from any available source, including systematiclibraries of natural and/or synthetic compounds. Test compounds may alsobe obtained by any of the numerous approaches in combinatorial librarymethods known in the art, including: biological libraries; peptoidlibraries (libraries of molecules having the functionalities ofpeptides, but with a novel, non-peptide backbone which are resistant toenzymatic degradation but which nevertheless remain bioactive; see,e.g., Zuckermann et al., 1994, J. Med. Chem. 37:2678-85); spatiallyaddressable parallel solid phase or solution phase libraries; syntheticlibrary methods requiring deconvolution; the ‘one-bead one-compound’library method; and synthetic library methods using affinitychromatography selection. The biological library and peptoid libraryapproaches are limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, 1997, Anticancer Drug Des.12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten,1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria and/orspores, (Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al, 1992,Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith, 1990,Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al,1990, Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol. Biol.222:301-310; Ladner, supra.).

The screening methods of the invention comprise contacting a cell, e.g.,a diseased cell, especially a prostate cancer cell, with a test compoundand determining the ability of the test compound to modulate theexpression and/or activity of filamin B, LY9, or keratin 19, optionallyin combination with PSA, in the cell. The expression and/or activity offilamin B, LY9, or keratin 19; optionally in combination with PSA, canbe determined using any methods known in the art, such as thosedescribed herein.

In another embodiment, the invention provides assays for screeningcandidate or test compounds which are substrates of a marker of theinvention or biologically active portions thereof. In yet anotherembodiment, the invention provides assays for screening candidate ortest compounds which bind to a marker of the invention or biologicallyactive portions thereof. Determining the ability of the test compound todirectly bind to a marker can be accomplished, for example, by anymethod known in the art.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent capable of modulatingthe expression and/or activity of a marker of the invention identifiedas described herein can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment (e.g., of prostatecancer) with such an agent. Alternatively, an agent identified asdescribed herein can be used in an animal model to determine themechanism of action of such an agent. Furthermore, this inventionpertains to uses of novel agents identified by the above-describedscreening assays for treatment as described above.

In certain embodiments, the screening methods are performed using cellscontained in a plurality of wells of a multi-well assay plate. Suchassay plates are commercially available, for example, from StratageneCorp. (La Jolla, Calif.) and Corning Inc. (Acton, Mass.) and include,for example, 48-well, 96-well, 384-well and 1536-well plates.

Reproducibility of the results may be tested by performing the analysismore than once with the same concentration of the same candidatecompound (for example, by incubating cells in more than one well of anassay plate). Additionally, since candidate compounds may be effectiveat varying concentrations depending on the nature of the compound andthe nature of its mechanism(s) of action, varying concentrations of thecandidate compound may be tested. Generally, candidate compoundconcentrations from 1 fM to about 10 mM are used for screening.Preferred screening concentrations are generally between about 10 pM andabout 100 μM.

The screening methods of the invention will provide “hits” or “leads,”i.e., compounds that possess a desired but not optimized biologicalactivity. Lead optimization performed on these compounds to fulfill allphysicochemical, pharmacokinetic, and toxicologic factors required forclinical usefulness may provide improved drug candidates. The presentinvention also encompasses these improved drug candidates and their useas therapeutics for modulating prostate cancer progression.

J. Kits/Panels

The invention also provides compositions and kits for diagnosing,prognosing, or monitoring a disease or disorder, recurrence of adisorder, or survival of a subject being treated for a disorder (e.g.,an abnormal prostate state, BPH, an oncologic disorder, e.g., prostatecancer). These kits include one or more of the following: a detectableantibody that specifically binds to a marker of the invention, adetectable antibody that specifically binds to a marker of theinvention, reagents for obtaining and/or preparing subject tissuesamples for staining, and instructions for use.

The invention also encompasses kits for detecting the presence of amarker protein or nucleic acid in a biological sample. Such kits can beused to determine if a subject is suffering from or is at increased riskof developing an abnormal prostate state. For example, the kit cancomprise a labeled compound or agent capable of detecting a markerprotein or nucleic acid in a biological sample and means for determiningthe amount of the protein or mRNA in the sample (e.g., an antibody whichbinds the protein or a fragment thereof, or an oligonucleotide probewhich binds to DNA or mRNA encoding the protein). Kits can also includeinstructions for use of the kit for practicing any of the methodsprovided herein or interpreting the results obtained using the kit basedon the teachings provided herein. The kits can also include reagents fordetection of a control protein in the sample not related to the abnormalprostate state, e.g., actin for tissue samples, albumin in blood orblood derived samples for normalization of the amount of the markerpresent in the sample. The kit can also include the purified marker fordetection for use as a control or for quantitation of the assayperformed with the kit.

Kits include a panel of reagents for use in a method to diagnoseprostate cancer in a subject (or to identify a subject predisposed todeveloping prostate cancer, etc.), the panel comprising at least twodetection reagents, wherein each detection reagent is specific for oneprostate cancer-specific protein, wherein said prostate cancer-specificproteins are selected from the prostate cancer-specific protein setsprovided herein.

For antibody-based kits, the kit can comprise, for example: (1) a firstantibody (e.g., attached to a solid support) which binds to a firstmarker protein; and, optionally, (2) a second, different antibody whichbinds to either the first marker protein or the first antibody and isconjugated to a detectable label. In certain embodiments, the kitincludes (1) a second antibody (e.g., attached to a solid support) whichbinds to a second marker protein; and, optionally, (2) a second,different antibody which binds to either the second marker protein orthe second antibody and is conjugated to a detectable label. The firstand second marker proteins are different. In an embodiment, the firstand second markers are markers of the invention, e.g., filamin A,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, filamin B, LY9, and PSA. In certain embodiments, neitherthe first marker nor the second marker is PSA. In certain embodiments,the kit comprises a third antibody which binds to a third marker proteinwhich is different from the first and second marker proteins, and asecond different antibody that binds to either the third marker proteinor the antibody that binds the third marker protein wherein the thirdmarker protein is different from the first and second marker proteins.

For oligonucleotide-based kits, the kit can comprise, for example: (1)an oligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a marker protein or (2) apair of primers useful for amplifying a marker nucleic acid molecule. Incertain embodiments, the kit can further include, for example: (1) anoligonucleotide, e.g., a second detectably labeled oligonucleotide,which hybridizes to a nucleic acid sequence encoding a second markerprotein or (2) a pair of primers useful for amplifying the second markernucleic acid molecule. The first and second markers are different. In anembodiment, the first and second markers are markers of the invention,e.g., filamin A, keratin 4, keratin 7, keratin 8, keratin 15, keratin18, keratin 19, tubulin-beta 3, filamin B, LY9, and PSA. In certainembodiments, neither the first marker nor the second marker is PSA. Incertain embodiments, the kit can further include, for example: (1) anoligonucleotide, e.g., a third detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a third marker protein or(2) a pair of primers useful for amplifying the third marker nucleicacid molecule wherein the third marker is different from the first andsecond markers. In certain embodiments, the kit includes a third primerspecific for each nucleic acid marker to allow for detection usingquantitative PCR methods.

For chromatography methods, the kit can include markers, includinglabeled markers, to permit detection and identification of one or moremarkers of the invention, e.g., filamin A, keratin 4, keratin 7, keratin8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, filamin B, LY9,and optionally PSA, by chromatography. In certain embodiments, kits forchromatography methods include compounds for derivatization of one ormore markers of the invention. In certain embodiments, kits forchromatography methods include columns for resolving the markers of themethod.

Reagents specific for detection of a marker of the invention, e.g.,filamin A, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, tubulin-beta 3, filamin B, LY9, and PSA, allow for detectionand quantitation of the marker in a complex mixture, e.g., serum, tissuesample. In certain embodiments, the reagents are species specific. Incertain embodiments, the reagents are not species specific. In certainembodiments, the reagents are isoform specific. In certain embodiments,the reagents are not isoform specific. In certain embodiments, thereagents detect total keratin 8, keratin 18, filamin B, PSA, or LY9.

In certain embodiments, the kits for the diagnosis, monitoring, orcharacterization of prostate cancer comprise at least one reagentspecific for the detection of the level of expression of at least onemarker selected from the group consisting of keratin 4, keratin 7,keratin 8, keratin 15, keratin 18, keratin 19, and tubulin-beta 3,filamin B, and LY9. In certain embodiments, the kits further compriseinstructions for the diagnosis, monitoring, or characterization ofprostate cancer based on the level of expression of the at least onemarker selected from the group consisting of keratin 4, keratin 7,keratin 8, keratin 15, keratin 18, keratin 19, and tubulin-beta 3,filamin B, and LY9. In certain embodiments, the kits further compriseinstructions to detect the level of PSA in a sample in which the atleast one marker selected from the group consisting of keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, andtubulin-beta 3, filamin B, and LY9 is detected. In certain embodiments,the kits further comprise at least one reagent for the specificdetection of PSA.

The invention provides kits comprising at least one reagent specific forthe detection of a level of expression of at least one marker selectedfrom the group consisting of keratin 4, keratin 7, keratin 8, keratin15, keratin 18, keratin 19, and tubulin-beta 3, filamin B, and LY9 andat least one reagent specific for the detection of a level of expressionof PSA.

In certain embodiments, the kits can also comprise, e.g., a bufferingagents, a preservative, a protein stabilizing agent, reaction buffers.The kit can further comprise components necessary for detecting thedetectable label (e.g., an enzyme or a substrate). The kit can alsocontain a control sample or a series of control samples which can beassayed and compared to the test sample. The controls can be controlserum samples or control samples of purified proteins or nucleic acids,as appropriate, with known levels of target markers. Each component ofthe kit can be enclosed within an individual container and all of thevarious containers can be within a single package, along withinstructions for interpreting the results of the assays performed usingthe kit.

The kits of the invention may optionally comprise additional componentsuseful for performing the methods of the invention.

The invention further provides panels of reagents for detection of oneor more prostate-related marker in a subject sample and at least onecontrol reagent. In certain embodiments, the control reagent is todetect the marker for detection in the biological sample wherein thepanel is provided with a control sample containing the marker for use asa positive control and optionally to quantitate the amount of markerpresent in the biological sample. In certain embodiments, the panelincludes a detection reagent for a maker not related to an abnormalprostate state that is known to be present or absent in the biologicalsample to provide a positive or negative control, respectively. Thepanel can be provided with reagents for detection of a control proteinin the sample not related to the abnormal prostate state, e.g., actinfor tissue samples, albumin in blood or blood derived samples fornormalization of the amount of the marker present in the sample. Thepanel can be provided with a purified marker for detection for use as acontrol or for quantitation of the assay performed with the panel.

In a preferred embodiment, the panel includes reagents for detection oftwo or more markers of the invention (e.g., 2, 3, 4, 5, 6, 7, 8, 9),preferably in conjunction with a control reagent. In the panel, eachmarker is detected by a reagent specific for that marker. In certainembodiments, the panel further includes a reagent for the detection ofPSA. In certain embodiments, the panel includes replicate wells, spots,or portions to allow for analysis of various dilutions (e.g., serialdilutions) of biological samples and control samples. In a preferredembodiment, the panel allows for quantitative detection of one or moremarkers of the invention.

In certain embodiments, the panel is a protein chip for detection of oneor more markers. In certain embodiments, the panel is an ELISA plate fordetection of one or more markers. In certain embodiments, the panel is aplate for quantitative PCR for detection of one or more markers.

In certain embodiments, the panel of detection reagents is provided on asingle device including a detection reagent for one or more markers ofthe invention and at least one control sample. In certain embodiments,the panel of detection reagents is provided on a single device includinga detection reagent for two or more markers of the invention and atleast one control sample. In certain embodiments, multiple panels forthe detection of different markers of the invention are provided with atleast one uniform control sample to facilitate comparison of resultsbetween panels.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references andpublished patents and patent applications cited throughout theapplication are hereby incorporated by reference.

EXAMPLES

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,GenBank Accession and Gene numbers, and published patents and patentapplications cited throughout the application are hereby incorporated byreference. Those skilled in the art will recognize that the inventionmay be practiced with variations on the disclosed structures, materials,compositions and methods, and such variations are regarded as within theambit of the invention.

Example 1: Identification of Keratins and Tubulin as Prostate CancerMarkers

Extracellular Keratins are known to influence the cell proliferation andmetastasis of epithelial derived prostate cancers. Androgen refractoryprostate cancers exhibit differential expression keratin 8 (K8) whencompared to normal tissue. Modulation and degradation of keratins is inturn mediated by mitochondrial generation of Reactive Oxygen Species(ROS). Despite these advances a systematic approach to understanding ofkeratins and other EC proteins in prostate cancer metastasis andproliferation is lacking.

An interrogative systems biology based discovery platform (i.e.,Interrogative Platform Technology or a.k.a. Interrogative Biology™)disclosed in WO2012119129 (incorporated herein by reference), and shownschematically in FIG. 1, provides new mechanistic insights intounderstanding mitochondrial role in behavior of prostate cancer cells.The discovery platform involves discovery across a hierarchy of systemsincluding in vitro human cell based models and human serum samples fromprostate cancer patients and downstream data integration andmathematical modeling employing an Artificial Intelligence (AI) basedinformatic module. For cellular models, androgen sensitive LnCAP cellline and metastatic, androgen refractory PC3 cell line were treated withubidecarenone (coenzyme Q10) in order to engage the mitochondrialmachinery. Proteomic signatures were captured using a 2D LC-MS orbitraptechnology. Total protein signatures were input to an AI basedinformatics module to generate causal protein networks (FIG. 2). Wet labassays that specifically measure mitochondrial ROS, ATP and caspase 3activation confirmed changes in intracellular levels of these markers.

As shown in FIG. 3, several novel protein causal interactions thatgovern induction of mitochondrial machinery by ubidecarenone in PC3cells were observed. Causal protein maps revealed association ofkeratins 8 and 15 in PC3 models and not LnCAP. The keratin 8/15association was lost upon treatment with ubidecarenone, and a directassociation of keratins 7 and 15 was established (FIG. 3).

These results suggest that a change in the interaction among keratins 7,8, and 15 is particularly useful in demonstrating a response totreatment or a change in prostate cancer status in a subject. Further,keratins 8 and 15 were differentially associated in the androgenrefractory, metastatic PC3 cell line and the androgen sensitive LnCAPcell line. This indicates that keratins 8 and 15 could be useful dodifferentiate between prostate cancer states, e.g., between androgensensitive and metastatic, androgen refractory prostate cancer.

An increase in the expression of keratin 19 in relation to prostatecancer was confirmed using a panel of serum samples from subjectssuffering from prostate cancer as compared to an appropriate matchedcontrol population. (See FIG. 2C and FIG. 3D).

Thus novel mechanistic insight into prostate cancer proliferation andmitochondrial role in modulating metastasis was gained with a novelchemical systems biology approach.

The results provided herein demonstrate that modulation of keratin andpotential causal association in androgen refractory prostate cancer wasinferred by the Platform technology. This provides a potentialmechanisms of keratin regulation in response to modulation ofmitochondrial function was deciphered by the Platform technology. Thus,novel drivers of cancer pathophysiology were validated in patient serumsamples.

Example 2: Identification of Filamin B as a Prostate Cancer Marker

An interrogative systems biology based discovery platform (i.e.,Interrogative Platform Technology or a.k.a. Interrogative Biology™) wasused to obtain mechanistic insights into understanding the mitochondrialrole in behavior of prostate cancer cells. The Platform technology,which is described in detail in WO2012119129, involves discovery acrossa hierarchy of systems including in vitro human cell based models andhuman serum samples from prostate cancer patients and downstream dataintegration and mathematical modeling employing an ArtificialIntelligence (AI) based informatics module.

The results provided in this Example demonstrate the modulation offilamin B and LY9, and the potential causal association in androgenrefractory prostate cancer that was inferred using the Platformtechnology. The Example provides potential mechanisms of filamin B andLY9 regulation in response to modulation of mitochondrial function thatwas deciphered by the Platform technology, and provides validation ofthe markers in patient serum samples.

Using the Platform methods, human prostate cancer cells PC3 (androgeninsensitive, metastatic) and LnCap (androgen sensitive) were modeled incancer microenvironments including hypoxia, reduced environments, andhyperglycemia and in presence of coenzyme Q10. Normal cells (humandermal fibroblasts (HDFa) and SV40 transformed human liver cells(THLE2)) were modeled under similar conditions mentioned above inExample 2. Proteomics of cellular proteins and proteins secreted in thesupernatant were carried out by LCMS. Data were input into the BayesianNetwork Inference (BNI) algorithms REFS™.

Causal associations between proteins were derived by the BNI.Differential network analysis was employed to tease out the hubs ofactivity in prostate cancer when compared to normal cells in normalmicroenviroments. Filamin B was identified as differential hub ofactivity in PC3 and not in LnCap and normal cells. That is, Filamin Bwas found to differ between androgen sensitive LnCAP cell line andmetastatic, androgen refractory PC3 cell line. This indicates thatFilamin B could be useful to differentiate between prostate cancerstates, e.g., between androgen sensitive and metastatic, androgenrefractory prostate cancer. The interaction matrix placing filamin B atthe center of an interaction hub is shown in FIG. 4. The interaction ofLY9 with filamin B is shown in FIG. 5.

Example 3: Validation of Filamin B as a Prostate Cancer Marker in HumanSamples

Having identified filamin B as a prostate cancer marker using theplatform technology, human serum samples from normal subjects andsubjects with prostate cancer were used to confirm filamin B as aprostate cancer marker.

Specifically, human serum samples were procured from a commercial vendorthat sources human serum. Twenty samples were from normal donors and 20samples were from patients diagnosed with prostate cancer. Prostatecancer samples were from patients with different prognosis andaggressiveness of cancers reported. Clinical characteristics of thesubjects are provided in the table.

Prostate Cancer Control Group Median Age 61 (47-86) 58 (45-72) EthnicityCaucasian 75% 85% African American 15% 10% Hispanic 10%  5% Tumor StageStage I 20% Stage II 35% Stage III  5% Stage IV 40%

Commercially available ELISA tests for filamin B and PSA were procuredfrom commercial source. The assays were performed using themanufacturer's instructions. The results from the assay are shown inFIG. 6. The results show the differential levels of FlnB and PSA inpatients with a diagnosis for prostate cancer as compared to controlsubjects without prostate cancer.

As shown, both filamin B and PSA levels were elevated in serum samplesfrom patients diagnosed with prostate cancer. The correlation betweenPSA and FlnB expression in serum samples is 0.20075, indicating arelatively low correlation between the variables. This demonstrates thatfilamin B and PSA are useful for the detection of prostate cancer indifferent subjects. These results demonstrate that filamin B is usefulfor the diagnosis of prostate cancer, and that filamin B is useful forimproving the detection of prostate cancer by PSA.

Example 4: Validation of LY9 as a Prostate Cancer Marker in HumanSamples

The same human serum samples used in Example 3 were further tested todetect the presence of LY9. A commercially available ELISA test for LY9was procured from commercial source. The assay was performed using themanufactures' instructions. The results from the assay are shown in FIG.7. The results show the differential levels of LY9 in patients with adiagnosis for prostate cancer as compared to control subjects withoutprostate cancer. As shown, samples from subjects with prostate cancerwere found to have higher levels of LY9 as compared to normal subjects.Results from assays of expression levels of both filamin B and LY9 inhuman serum with results expressed as ng/ml of protein are shown in FIG.8.

Example 5: Analysis of Filamin B Levels Improves the Detection ofProstate Cancer as Compared to PSA Alone

Having demonstrated that the level of filamin B is increased in theserum of subjects with prostate cancer, the results were analyzed inconjunction with the study of PSA levels in the same samples todetermine if the predictive value of filamin B and PSA together wasbetter than either of the markers alone. Receiver operatingcharacteristic (ROC) curve analysis of sensitivity and false positiverate (FPR) of PSA, filamin B, and the combination of PSA and filamin Bwas generated. The curves and the area under the curve (AUC) values areshown in FIGS. 9A and B. The goal of this analysis was to gauge thepredictive power of the test independent of a specific cut-off. Whenusing an ROC analysis, a test that provides perfect discrimination oraccuracy between normal and disease states would have AUC=1, whereas avery poor test that provides no better discrimination than random chancewould have AUC=0.5

As demonstrated by the analysis, filamin B alone performs very well andmost importantly somewhat orthogonal to PSA. PSA is reported to have avery high false positive rate, e.g., about 75% (as reported in,Gilligan, “The new data on prostate cancer screening: What should we donow?,” Cleveland Clin. J. Med. 76: 446-448, 2009, incorporated herein byreference). That is, it has a high sensitivity and low specificity. Inthe specific study presented, the AUC for FLNB is lower than that forPSA. However, the correlation level of 0.20075 determined in Example 3,indicates a relatively low correlation between the variables. That is,subjects identified as having an elevated filamin B level did notnecessarily have a high PSA level, and the reverse was also true,suggesting that the markers in combination can provide a more predictivetest than either marker alone.

This was confirmed in the ROC analysis. As shown, the combination of PSAand filamin B was found to have a higher AUC indicating betterdiscrimination of the test than PSA alone, and to be more predictivethan either of the markers alone. The combination of PSA and filamin Bis a very good predictor of prostate cancer and provides a drasticincrease over the PSA test specificity, which is the primary problemwith the test.

Example 6: Analysis of Filamin B, LY9, and PSA Levels Together Improvesthe Detection of Prostate Cancer as Compared to any Marker Alone

Having demonstrated that each of filamin B, LY9, and PSA are allelevated in serum samples from subjects with prostate cancer, the ROCcurve analysis was performed comparing each of the three markersindividually to the combination of all three markers using a linearscoring function, and comparing the combination of filamin B and LY9,and the combination of filamin B and PSA, against the combination of allthree markers using a non-linear scoring function to determine whichcombinations of the markers were more effective than each single markerfor the detection of prostate cancer in a subject. As shown, thecombination of all three markers was more predictive than any of themarkers alone (FIG. 10A). The combination of filamin B with PSA, eitherwith or without LY9, was more predictive than the combination of filaminB with LY9 (FIG. 10B). Additional samples can be analyzed to furtherrefine the results. The AUC results are summarized in the table.

Marker AUC LY9 0.85 FLNB 0.78 PSA 0.87 LY9 + FLNB + PSA 0.98

Example 7: Stratification of Subjects with Prostate Cancer Using Keratin4, Keratin 7, Keratin 8, Keratin 15, Keratin 18, Keratin 19,Tubulin-beta 3

As demonstrated in Examples 3 and 4 respectively, filamin B levels andLY9 levels can be used to distinguish subjects who are or are notsuffering from prostate cancer. Further, as demonstrated in Examples 5and 6, the analysis of both filamin B and PSA, optionally further incombination with LY9, is more sensitive than an analysis based on eithermarker alone. The markers keratin 4, keratin 7, keratin 8, keratin 15,keratin 18, keratin 19, and tubulin-beta 3 are similarly analyzed, asdescribed in Examples 3-6, in human samples.

A series of subject samples are obtained from an appropriate source,e.g., a commercial source, wherein the samples were obtained fromsubjects with different stages of prostate cancer, e.g., aggressiveprostate cancer, androgen sensitive, androgen insensitive, metastatic;or from subjects not suffering from prostate cancer, e.g., subjects withnormal prostate or subjects with BPH. The samples are analyzed for theexpression level of at least one of keratin 4, keratin 7, keratin 8,keratin 15, keratin 18, keratin 19, tubulin-beta 3, preferably at leastone of keratin 7, keratin 15, and keratin 19; and optionally further atleast one of filamin B, LY9, and PSA. The level of the expression of themakers, alone and in various combinations, correlate with the presenceor absence of disease, and with the severity of prostate cancer. Forexample, an increase in the expression level of one or more of keratin19, filamin B, LY9, and PSA, as compared to a normal sample from asubject not suffering from prostate cancer, is indicative of prostatecancer in the subject. Expression levels of keratins 7, 8, and 15 mayalso be particularly useful in the stratification of subjects withprostate cancer.

Example 8: Monitoring of Prostate Cancer Treatment Using Keratin 4,Keratin 7, Keratin 8, Keratin 15, Keratin 18, Keratin 19, Tubulin-Beta 3

At the time of diagnosis with prostate cancer, subjects are invited toparticipate in a trial. A subject sample, e.g., blood, is obtained.Periodically, throughout the monitoring, watchful waiting, or activetreatment of the subject, e.g., chemotherapy, radiation therapy,surgery, hormone therapy, a new subject sample is obtained. At the endof the study, all subject samples are tested for the expression level ofat least one of keratin 4, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, tubulin-beta 3, preferably at least one of keratin 7,keratin 15, and keratin 19; and optionally further at least one offilamin B, LY9, and PSA. The subject samples are matched to the medicalrecords of the subjects to correlate marker levels with prostate cancerstatus at the time of diagnosis, rate of progression of disease,response of subjects to one or more interventions, and transitionsbetween androgen dependent and independent status. An increase in theexpression level of one or more of keratin 19, filamin B, LY9, and PSA,as compared to a normal sample from a subject not suffering fromprostate cancer, is indicative of prostate cancer in the subject.Expression levels of keratins 7, 8, and 15 may also be particularlyuseful in the diagnosis and monitoring of subjects with prostate cancer.

Example 9: Detection and Monitoring of Prostate Cancer Using Keratin 4,Keratin 7, Keratin 8, Keratin 15, Keratin 18, Keratin 19, Tubulin-Beta 3

Despite its limitations, including a positive predictive value of only25-40%, PSA remains the only generally accepted biomarker for prostatecancer. Moreover, as prostate cancer is most commonly a slow growingtumor in men of advanced age, treatment of the cancer may do more harmto the subject than the tumor itself would. Therefore, the teststogether for the expression level of at least one of keratin 4, keratin7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3,preferably at least one of keratin 7, keratin 15, and keratin 19; andoptionally further at least one of filamin B, LY9, and PSA are used forthe detection an monitoring of prostate cancer. The level of theexpression of the makers, alone and in various combinations are used indetection, including in routine, preventative, screening methods in menhaving an increased risk of prostate cancer (e.g., increased age, familyhistory, race, etc.) or in monitoring of subjects diagnosed withprostate cancer prior to or during treatment may be useful to betteridentify subjects in need of further, potentially more invasive,diagnostic tests, e.g., prostate exam or biopsy, digital rectal exam; ormore aggressive treatment. Detection of levels of expression of themarkers, or various combinations thereof, may also be indicative of agood or poor response to a specific treatment regimen prior to changesin other signs or symptoms, e.g., loss of tumor response to hormonetherapy.

In routine screening methods for prostate cancer, a serum sample from asubject is tested for the level of expression of at least one of keratin4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19,tubulin-beta 3, preferably at least one of keratin 7, keratin 15, andkeratin 19; and optionally further at least one of filamin B, LY9, andPSA. The levels are compared to one or more appropriate controls, e.g.,other normal subjects, subjects with prostate cancer. Detection of anabnormal level of one or more of at least one of keratin 4, keratin 7,keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3,preferably at least one of keratin 7, keratin 8, keratin 15, and keratin19; indicates that the subject should be considered for further testsfor the presence of prostate cancer. Changes in the level of at leastone of keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin19, tubulin-beta 3, preferably at least one of keratin 7, keratin 8,keratin 15, and keratin 19, in the subject may be more indicative of achange in prostate cancer status than comparison to a populationcontrol.

In determining a therapeutic regimen for a subject with prostate cancernot yet being actively treated for prostate cancer (i.e., watchfulwaiting) can be tested at regular intervals to determine if there is achange in the level of expression of at least one of keratin 4, keratin7, keratin 8, keratin 15, keratin 18, keratin 19, tubulin-beta 3,preferably at least one of keratin 7, keratin 15, and keratin 19; andoptionally further at least one of filamin B, LY9, and PSA. Anmodulation in the level of at least one of keratin 4, keratin 7, keratin8, keratin 15, keratin 18, keratin 19, tubulin-beta 3, preferably atleast one of keratin 7, keratin 8, keratin 15, and keratin 19; andoptionally further at least one of filamin B, LY9, and PSA indicatesthat the subject should be considered for further tests to monitor theprostate cancer and more active therapeutic interventions should beconsidered.

In a subject undergoing treatment for prostate cancer (e.g., hormonetherapy, chemotherapy, radiation therapy, surgery) is tested prior tothe initiation of the treatment and during and/or after the treatment todetermine if the treatment results in a decrease in the level ofexpression of at least one of keratin 4, keratin 7, keratin 8, keratin15, keratin 18, keratin 19, tubulin-beta 3, preferably at least one ofkeratin 7, keratin 15, and keratin 19; and optionally further at leastone of filamin B, LY9, and PSA. A decrease in the level of keratin 19,filamin B, LY9, or PSA is indicative of response to treatment.Expression levels of keratins 7, 8, and 15 may also be particularlyuseful in the diagnosis and monitoring of subjects with prostate cancer.

Example 10: Stratification of Subjects with Prostate Cancer UsingFilamin B, PSA, or LY9

As demonstrated in Examples 3 and 4 respectively, filamin B levels andLY9 levels can be used to distinguish subjects who are or are notsuffering from prostate cancer. Further, as demonstrated in Examples 5and 6, the analysis of both filamin B and PSA, optionally further incombination with LY9, is more sensitive than an analysis based on eithermarker alone.

A series of subject samples are obtained from an appropriate source,e.g., a commercial source, wherein the samples were obtained fromsubjects with different stages of prostate cancer, e.g., aggressiveprostate cancer, androgen sensitive, androgen insensitive, metastatic;or from subjects not suffering from prostate cancer, e.g., subjects withnormal prostate or subjects with BPH. The samples are analyzed for theexpression level of filamin B and PSA, and optionally the level of LY9,and further with one or more of keratin 4, keratin 7, keratin 8, keratin15, keratin 18, keratin 19, and tubulin-beta 3, especially keratin 19.The level of filamin B, LY9, and PSA, alone and in various combinations,optionally with other markers, e.g., keratin 4, keratin 7, keratin 8,keratin 15, keratin 18, keratin 19, and tubulin-beta 3, especiallykeratin 19, correlate with the presence or absence of disease, and withthe severity of prostate cancer.

Example 11: Monitoring of Prostate Cancer Treatment Using Filamin B,PSA, or LY9

At the time of diagnosis with prostate cancer, subjects are invited toparticipate in a trial. A subject sample, e.g., blood, is obtained.Periodically, throughout the monitoring, watchful waiting, or activetreatment of the subject, e.g., chemotherapy, radiation therapy,surgery, hormone therapy, a new subject sample is obtained. At the endof the study, all subject samples are tested for the level of filamin B,PSA, and optionally in further combination with one or more of LY9,keratin 4, keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, andtubulin-beta 3. The subject samples are matched to the medical recordsof the subjects to correlate filamin B, PSA, LY9, keratin 4, keratin 7,keratin 8, keratin 15, keratin 18, keratin 19, or tubulin-beta 3 levels,as appropriate, with prostate cancer status at the time of diagnosis,rate of progression of disease, response of subjects to one or moreinterventions, and transitions between androgen dependent andindependent status.

Example 12: Detection and Monitoring of Prostate Cancer Using Filamin B,PSA, or LY9

Despite its limitations, including a positive predictive value of only25-40%, PSA remains the only generally accepted biomarker for prostatecancer. Moreover, as prostate cancer is most commonly a slow growingtumor in men of advanced age, treatment of the cancer may do more harmto the subject than the tumor itself would. As demonstrated herein,there is a low correlation between elevated levels of filamin B and PSAin subjects with prostate cancer. Further, elevated levels of LY9 havebeen demonstrated to be associated with prostate cancer. Therefore, thetests together, particularly filamin B and PSA, optionally incombination with one or more of LY9, keratin 4, keratin 7, keratin 8,keratin 15, keratin 18, keratin 19, and tubulin-beta 3, especiallykeratin 19, in detection, including in routine, preventative, screeningmethods in men having an increased risk of prostate cancer (e.g.,increased age, family history, race, etc.) or in monitoring of subjectsdiagnosed with prostate cancer prior to or during treatment may beuseful to better identify subjects in need of further, potentially moreinvasive, diagnostic tests, e.g., prostate exam or biopsy, digitalrectal exam; or more aggressive treatment. Detection of levels ofexpression of filamin B, PSA, LY9 keratin 4, keratin 7, keratin 8,keratin 15, keratin 18, keratin 19, and tubulin-beta 3, especiallykeratin 19, may also be indicative of a good or poor response to aspecific treatment regimen prior to changes in other signs or symptoms,e.g., loss of tumor response to hormone therapy.

In routine screening methods for prostate cancer, a serum sample from asubject is tested for the level of expression of both filamin B and PSA,and optionally one or more of LY9, keratin 4, keratin 7, keratin 8,keratin 15, keratin 18, keratin 19, and tubulin-beta 3, especiallykeratin 19. The levels are compared to one or more appropriate controls,e.g., other normal subjects, subjects with prostate cancer. Detection ofan abnormal level of one or more of filamin B, PSA, LY9, keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, andtubulin-beta 3, especially keratin 19 indicates that the subject shouldbe considered for further tests for the presence of prostate cancer.Changes in the level of filamin B, optionally in combination with one ormore of PSA, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin18, keratin 19, or tubulin-beta 3, especially keratin 19 with PSA in thesubject may be more indicative of a change in prostate cancer statusthan comparison to a population control.

In determining a therapeutic regimen for a subject with prostate cancernot yet being actively treated for prostate cancer (i.e., watchfulwaiting) can be tested at regular intervals to determine if there is achange in the level of expression of filamin B, PSA, LY9 keratin 4,keratin 7, keratin 8, keratin 15, keratin 18, keratin 19, andtubulin-beta 3. An increase in the level of filamin B, PSA, keratin 19,or LY9 indicates that the subject should be considered for further teststo monitor the prostate cancer and more active therapeutic interventionsshould be considered.

In a subject undergoing treatment for prostate cancer (e.g., hormonetherapy, chemotherapy, radiation therapy, surgery) is tested prior tothe initiation of the treatment and during and/or after the treatment todetermine if the treatment results in a change in the level ofexpression of one or more of filamin B, PSA, LY9, keratin 4, keratin 7,keratin 8, keratin 15, keratin 18, keratin 19, and tubulin-beta 3. Adecrease in the level of filamin B, PSA, keratin 19, or LY9 isindicative of response to treatment.

Example 13: Detection of Filamin a and Keratin 19 by ELISA in HumanSerum from Patients with and without Prostate Cancer

ELISA assays were conducted on commercially available human serumsamples to detect the levels of filamin A and keratin 19 in samples frompatients with and without prostate cancer.

The commercial human serum samples used for the filamin A ELISA areshown in FIG. 11, the data/annotation of which is publicly available atthe Asterand and Bioreclamation websites. The commercial human serumsamples used for the keratin 19 ELISA are shown in FIG. 12, thedata/annotation of which is publicly available at the Asterand andBioreclamation websites.

For the filamin A test, the ELISA was conducted in accordance with theHuman Filamin A (FLNa) ELISA kit, Catalog No. CSB-EL008724HU, which iscommercially and publicly available from CEDARLANE (Cusabio), thecontents of which are incorporated herein by reference.

For the keratin 19 test, the ELISA was conducted in accordance with theHuman Keratin-19 (KRT19) ELISA Kit, Catalog No. E91239HU, which iscommercially and publicly available from CEDARLANE (Cusabio), thecontents of which are incorporated herein by reference.

FIG. 13 shows the filamin A protein levels in serum from patients withand without prostate cancer as determined by ELISA.

FIG. 14. shows the keratin 19 protein levels in serum from patients withand without prostate cancer as determined by ELISA.

Example 14: Filamin a and Keratin 19 Differentiate Between Patients withProstate Cancer and Normal Individuals

The inventors have developed in-vitro models of prostate cancer based ona number of cancer and normal cell lines including the androgenindependent PC3 and the androgen dependent LNCaP prostate cancer celllines. Key regulatory nodes inferred by Berg Interrogative Biology™ wereselected for a proof-of-concept validation in human serum as biomarkersof prostate cancer.

The initial two biomarkers FLNB and LY9 showed predictive power todifferentiate between normal serum and serum from prostate cancerpatients. This report reviews statistical characteristics of additionalbiomarkers, FLNA, FLNC, KRT18 and KRT19.

Statistical Performance of Candidate Markers: Bioreclamation Sample Set#1:

Human serum samples from normal individuals and individuals withprostate cancer were acquired from a commercial source, as per Example13. The panel of biomarkers was measured by commercially available ELISAkits, as per Example 13.

FIG. 15 demonstrates the performance of the candidate markers in the setof 20 normal and 20 prostate cancer samples. FLN-A showed predictivepower to differentiate between normal and cancer patients, whereas FLN-Cdid not show any positive trend. The combination panel of FLN-A andFLN-C did not improve on the performance of FLN-A alone.

Statistical Performance of Candidate Markers: Bioreclamation andAsterand Sample Set #2:

Human serum samples from normal individuals and individuals withprostate cancer were acquired from two commercial sources, as perExample 13. The panel of biomarkers was measured by commerciallyavailable ELISA kits, as per Example 13.

FIG. 16 demonstrates the performance of the candidate markers in the setof 20 normal and 20 prostate cancer samples. KRT19 showed predictivepower to differentiate between normal and cancer patients. KRT18 did notshow any differentiation between normal and cancer specimens. Thecombination panel of KRT18 and KRT19 did not improve on the performanceof KRT19 alone.

Conclusion:

Based on the results of this proof-of-concept study, KRT19 and FLNA havethe potential to differentiate between patients with prostate cancer andnormal individuals. These two biomarkers may be evaluated in a largerclinical validation study by either a commercial or in-house developedELISA assay. The results above demonstrate that FLNa and KRT19 arestatistically significant biomarkers of prostate cancer and thereforemay be a significant improvement over the current screening tools.

Example 15: Filamin a, Filamin B, Keratin 19, and Age DifferentiateBetween Patients with Prostate Cancer and Normal Individuals

To study whether the Prostate Cancer Panel test is equivalent, better,or weaker than PSA in screening for prostate cancer, we studied theabove 4 comparisons with 4 different MVI (Multivariate Index) models tobest study the utility of the panel.

Samples were divided as training/verification set and validation set.The verification set consisting of 332 samples was used to develop themodels. The validation set of 171 samples were used to test the model.

The annotations of the validation samples were blinded. Probability unitlinear regression models were estimated using the verification part ofthe sample set. Regression models were created to classify samples fromthe patients with diagnosed prostate cancer, with high Gleason Score (>6and >7), and with benign prostate hyperplasia. Prostate cancer panelpredictive algorithms were implemented based on the regression modelsand the probability cut-offs selected to achieve a certain level of testsensitivity or specificity. Each predictive algorithm was validated onthe left-out validation sample set.

The biomarkers in the panel are:

1) Filamin—A

2) Filamin—B

3) Keratin—19

4) Age was used as a continuous predictor variable.

Using the panel in various combinations along with clinical information,the predictive power in screening for prostate cancer compared to PSAalone was tested. The specific study objectives were:

-   -   1) Determine the predictive power and the utility of the        Biomarker panel to differentiate between samples from patients        with and without prostate cancer.    -   2) Determine the predictive power of the Biomarker panel to        differentiate between samples from patients with HIGH Gleason        Score (8-10), INTERMEDIATE Gleason Score (7), and LOW Gleason        Score (6) prostate cancer.    -   3) Determine the predictive power of the Biomarker panel to        identify samples from patients with prostate cancer but low PSA        (less than 4 ng/ml) concentration.    -   4) Determine the predictive power of the Biomarker panel to        differentiate between samples from patients with prostate cancer        and benign prostatic hyperplasia.

The terminology used for categories of cancer is:

Super High=Gleason Score 8 and above.

High=Gleason Score 7 and above

Low=Gleason score 6

Else=All other samples when a certain specified category is beingcompared with the rest of the samples.

Study Design

A retrospective and clinically annotated sample set from Mt. SinaiHospital, Toronto, Canada was obtained. The sample matrix is serum andis frozen since its collection and processing.

This is a clinically annotated sample set with 662 samples from thepatients at a Prostate Center. The patients were all comers for prostategland biopsy. 120 samples from this set were used as a proof of conceptset for the proposed Prostate Cancer Panel. The remaining samples weresorted as per the volume available. Anything short of 350 ul wasexcluded and the samples were then segregated in 2:1 ratio andidentified as ‘Verification’ set and ‘Validation’ set.

All samples, irrespective of the verification or validation set, wereanalyzed using the prostate Cancer Panel in the CLIA certifiedlaboratory. The laboratory was blinded to the sample's designation of‘verification’ or ‘validation’ set.

The samples were randomized and separated as ‘verification’ and‘validation’ set.

Participant Recruitment and Sampling

Serum samples were collected from 662 male patients. All patients werereferred to a Prostate Center for a prostatic biopsy. These patientswere suspicious for prostate cancer based on either clinical symptoms,digital-rectal examination, or, more frequently, due to a serum PSAelevation (beyond 3 ng/ml). The patients were consented to participatein this study by a personal interview and blood was drawn before anyclinical manipulations or prostatic biopsy.

To establish the final diagnosis, the pathology report was examined by aclinical associate and patients were categorized as prostate cancer withGleason score assigned (n=311), benign conditions (n=122), atypicalsmall acini proliferation (n=26), prostatic intraepithelial neoplasia(n=69), benign prostatic hyperplasia (n=60), inflammation (n=58),microfocus adenocarcinoma (n=16).

Sample Collection Protocol

-   -   Venous blood was collected in SST tube, 5 mL from BD    -   Tubes were kept vertically at room temperature for ½-1 hour,        allowing the blood to clot.    -   Samples were centrifuged at 2000 RCF for 10 minutes at 4° C.    -   The supernatant serum was collected, aliquoted into 0.5 ml        sterile screw-capped tubes, and labeled with the appropriate        Identification Number.    -   Samples were immediately stored at −80° C.

Data Collection

-   -   There are 332 samples in the training and 171 samples in the        validation dataset. Samples were randomized to make two groups.    -   No. of total samples=332    -   No. of Gleason above 8=19    -   No. of samples with Gleason 6=103    -   No. of samples with No-Cancer=159    -   No. of BPH samples=23

Study Population

The samples were transferred on dry ice and were stored at −80° C. inthe CLIA certified laboratory.

The samples are from a retrospective sample set with the followingannotation:

-   -   PSA test values    -   Diagnosis    -   Patient age    -   Reason for biopsy    -   Total biopsy cores, staging, and grading (Gleason score)    -   Number of positive cores    -   Tumor location

Reference Standard and Rationale

-   -   The PSA test is the reference standard against which the outcome        of the Panel was compared.    -   This retrospective sample set has documented PSA values which        were used in data analysis.    -   This set also had documented biopsy results with Gleason scores.

Validated Assays

All samples were tested using the assays described below:

-   -   FLN—A ELISA: which measures FLN—A in serum and plasma by ELISA        in the range of 3.125-200 ng/ml    -   FLN—B ELISA which measures concentration of FLN-B in human serum        and plasma by ELISA in the range of 0.156-10 pM    -   Keratin—19 ELISA which measures soluble cytokeratin 19 fragments        in human serum    -   FLN—A IP MRM which measures the concentration of FLN A peptides        from human serum using Immunoprecipitation and LC-MS/MS in the        range of 125 pg/ml-2000 pg/ml for P2, 250 pg/ml—6000 pg/ml for        P3 and 1125 pg/ml—36000 pg/ml for P4.

Definition and Rationale of Units

-   -   PSA is reported in units of ng/ml. The values are taken from the        annotations of the sample set.    -   Filamin A ELISA:ng/ml    -   Filamin A Peptide IP—MRM:pg/ml    -   Keratin—19 fragment ELISA:ng/ml    -   Filamin—B ELISA; picomole, pM

Results

The models generated from the verification set samples generated thefollowing AUC data in Table 1 and the predictive power data in Table 2.

TABLE 1 AUC summary for all dataset (Peptide in Log scale) Super HighGleason CA vs Others CA vs BPH High Gleason vs. Low High Gleason vs.Others vs. Others Biomarker AUC Biomarker AUC Biomarker AUC BiomarkerAUC Biomarker AUC Individual PSA 0.569 PSA 0.564 PSA 0.619 PSA 0.631 PSA0.67 Age 0.62 Age 0.621 Age 0.622 Age 0.662 Age 0.7 FLNA 0.574 FLNA0.696 FLNA 0.515 FLNA 0.56 FLNA 0.542 FLNB 0.51 FLNB 0.516 FLNB 05.28FLNB 0.515 FLNB 0.515 Krt19 0.519 Krt19 0.618 Krt19 0.573 Krt19 0.567Krt19 0.574 P2-1 0.55 P2-1 0.636 P2-1 0.527 P2-1 0.551 P2-1 0.528 P2-20.502 P2-2 0.582 P2-2 0.528 P2-2 0.552 P2-2 0.53 P3-1 0.51 P3-1 0.524P3-1 0.547 P3-1 0.529 P3-1 0.5 P4-2 0.522 P4-2 0.595 P4-2 0.551 P4-20.555 P4-2 0.578 Two Combined Age & 0.63 FLNA & 0.717 Age & 0.659 Age &0.696 Age & 0.765 (top) FLNB KRT19 PSA PSA PSA Three Combined Age &0.635 FLNA & 0.735 PSA & 0.685 PSA & 0.717 PSA & 0.804 (top) FLNB KRT19& Age & Age & Age & & PSA Age FLNB FLNB FLNB Four Combined Age & 0.637FLNA & 0.746 PSA & 0.693 PSA & 0.723 PSA & 0.81 (top) FLNA & KRT19 & Age& Age & Age & P2-1 & P2-1 & FLNB & FLNP & FLNB & P2-2 Age P3-1 P3-1 P2-2FLAN & 0.746 PSA & 0.81 KRT19 & Age & Age & FLNB & P4-2 P2-2 FiveCombined Age & 0.644 FLNA & 0.764 PSA & 0.694 No improvement PSA & 0.811(top) FLNA & KRT19 & Age & Age & PSA & Age & FLNA & FLNB & P2-2 & P2-1 &P3-1 & P2-2 & P2-1 P3-1 KRT19 P2-1

TABLE 2 Summary of Predictive Power analysis of Verification Samples CutSensi- Speci- Goal off tivity ficity PPV NPV Differentiate betweenCancer and Non Cancer samples Verification Set 0.4455 0.7712 0.33800.5566 0.5783 Gleason Score 8 and above Vs. Other samples VerificationSet 0.01997 1.0000 0.2482 0.0752 1.0000 Gleason Score 7 and above Vs.Other samples Verification Set 0.1603 0.8136 0.4915 0.2857 0.9134Prostate Cancer Vs. BPH samples Verification Set 0.8758 0.8035 0.60870.9392 0.2917

The predictive power and the utility of the Biomarker panel todifferentiate between samples from patients with and without prostatecancer is shown below:

Differentiate between Cancer and Goal Non Cancer Cut off SensitivitySpecificity PPV NPV Comments Verification Set 0.4455 0.7712 0.33800.5566 0.5783 Validation Set 0.4455 0.8442 0.3580 0.5556 0.7073 p valuesignificant for Odds Ratio

Verification Set

This model was trained to match the sensitivity of PSA=4 cutoff and testthe specificity of the above-identified biomarker panel compared to thePSA test. FIG. 18 depicts PCA vs. Else: Sensitivity match PSA with acutoff of 0.4455.

Validation Set

A boxplot of the predicted probability and the accuracy analysis areshown in FIGS. 19 and 20, respectively.

Next, the predictive power of the biomarker panel to differentiatebetween samples from patients with SUPER HIGH Gleason Score (8-10), HIGHGleason Score (7 and above), and LOW Gleason Score (6) prostate cancerwas determined.

Goal Cut off Sensitivity Specificity PPV NPV Comments Gleason Score 8and above Vs. Other samples Verification Set 0.01997 1.0000 0.24820.0752 1.0000 Validation Set 0.01997 0.7500 0.2583 0.0509 0.9512 GleasonScore 7 and above Vs. Other samples Verification Set 0.1603 0.81360.4915 0.2857 0.9134 Validation Set 0.1603 0.7000 0.5116 0.2500 0.8800 pvalue significant for Odds Ratio

Verification Set

The model was trained to give a Sensitivity> or =0.95. As per thismodel, the cut off generated was 0.01997. See FIG. 21.

Validation Set

A boxplot of the predicted probability and the accuracy analysis areshown in FIG. 22 and FIG. 23, respectively.

Next, the predictive power of the biomarker panel to differentiatebetween samples from patients with a HIGH Gleason Score versusEverything Else was determined.

Verification Set

The model was trained to have sensitivity of greater than or equal to0.8. The outcome was a cut off value for the Biomarker panel which was0.1603. See FIG. 24.

Validation Set

A boxplot of the predicted probability and the accuracy analysis areshown in FIG. 25 and FIG. 26, respectively.

Next, the predictive power of the biomarker panel to identify samplesfrom patients with prostate cancer but low PSA (less than 4 ng/ml)concentration was determined. For this test, verification samples thathad cancer were differentiated as low PSA (less than 4 ng/ml) and highPSA (greater than 4 ng/ml). Thus, the PSA AUC was equal to 1. Next, thelow PSA and high PSA were predicted using the above-identified biomarkerpanel. The Table, below, depicts the AUC Summary for High PSA cancerversus Low PSA cancer.

AUC Summary for High PSA Cancer Vs. Low PSA Cancer

High PSA CA versus LPSA CA Biomarker AUC Age 0.598 FLNA 0.608 FLNB 0.517KRT19 0.517 P3-1 0.532 P4-2 0.506 Two Combined (top) FLNA & Age 0.664Three Combined (top) FLNA & Age & P4-2 0.669 Four Combined (top) Noimprovement Five Combined (top) No improvment

Next, the predictive power of the biomarker panel to differentiatebetween samples from patients with prostate cancer and benign prostatichyperplasia was determined. See Table, below.

Prostate Cancer Goal Vs. BPH Cut off Sensitivity Specificity PPV NPVComments Verification Set 0.8758 0.8035 0.6087 0.9392 0.2917 ValidationSet 0.8758 0.6790 0.3636 0.8871 0.1333

Verification Set

The biomarker panel was set to get sensitivity greater than or equal to0.8. The cut off generated by this model was 0.8758. See FIG. 27.

Validation Set

A boxplot of the predicted probability and the accuracy analysis areshown in FIG. 28 and FIG. 29, respectively.

Conclusion

The verification and validation study indicates utility in four clinicalapplications of the PCA panel. The developed model significantlyimproved the ability to discriminate less aggressive forms from moreaggressive forms. The AUC for discriminating samples taken from patientswith Gleason Scores 8 and higher was 0.8 (versus 0.67 for PSA alone).The negative predictive value was very high for this model, if thepatient value was below the model cutoff, the probability of the patientbeing disease free was 95%.

The model also performed well in discriminating patients with BPH versusprostate cancer. A patient value greater than the model cut-off wasassociated with an approximately 90% probability of the patient havingprostate cancer instead of BPH. The AUC for this model was 0.75 (versus0.56 for PSA alone).

The capabilities to discriminate samples from patients with prostatecancer from those without prostate cancer had predictive values in the55-70 percentiles. However, AUC's were improved compared to the use ofPSA alone.

Summary of Predictive Power analysis of Verification/Validation Samples

Goal Cut off Sensitivity Specificity PPV NPV Comments Differentiatebetween Cancer and Non Cancer Verification Set 0.4455 0.7712 0.33800.5566 0.5783 Validation Set 0.4455 0.8442 0.3580 0.5556 0.7073 p valuesignificant for Odds Ratio Gleason Score 8 and above Vs. Other samplesVerification Set 0.01997 1.0000 0.2482 0.0752 1.0000 Validation Set0.01997 0.7500 0.2583 0.0509 0.9512 Gleason Score 7 and above Vs. Othersamples Verification Set 0.1603 0.8136 0.4915 0.2857 0.9134 ValidationSet 0.1603 0.7000 0.5116 0.2500 0.8800 p value significant for OddsRatio Prostate Cancer Vs. BPH Verification Set 0.8758 0.8035 0.60870.9392 0.2917 Validation Set 0.8758 0.6790 0.3636 0.8871 0.1333

Example 16: Identification and Validation of Novel Prostate CancerBiomarkers

Prostate cancer is the most frequent cancer diagnosis among men and thesecond leading cause of cancer-related death. Despite the widespread useof digital rectal exam (DRE) and blood-based screening ofprostate-specific antigen (PSA) for prostate cancer screening, there aresignificant limitations in their specificity and prognostic value.Biomarkers which distinguish i) PSA-low prostate cancer from benignprostatic hyperplasia (BPH) and (ii) indolent versus aggressive diseasecourse represent unmet clinical needs. Experimentally, a panel ofprostate cancer cell lines and non-tumorigenic, human primary cells wereexposed to in vitro conditions designed to stimulate poor oxygenation,low pH, diminished nutrient microenvironments, and metabolicperturbations (24-48 h) followed by iTRAQ proteomic analysis of celllysates. Using an Interrogative Biology platform, proteomic data werethen subjected to Bayesian network learning to map molecularinteractions, with cytoskeletal and scaffolding proteins Filamin A(FLNA), Filamin B (FLNB), and Keratin 19 (KRT19) identified as candidateprostate cancer biomarkers.

To validate biomarker expression, mRNA and protein was quantified in apanel of primary human prostate epithelial cells (HPrEC) andandrogen-sensitive (LnCAP) or refractory (DU145, PC-3) prostate cancercells, and each was differentially detected in one or more prostatecancer cell lines compared to HPrEC (FIGS. 30 and 31). Specifically,basal expression of FLNA, FLNB, and KRT19 in prostate cancer cells invitro was assessed. mRNA and cell lysates were prepared from HPrEC,LnCAP, DU145, and PC-3 cells. Expression of FLNA, FLNB, and KRT19 wasassessed by quantitative RT-PCR and normalized to TBP. Cell lysates wereresolved by SDS-PAGE and probed with antibodies specific for FLNA, FLNB,and KRT19. Representative images are shown in FIGS. 30 and 31.Densitometric analysis for FLNA, FLNB, and KRT19 are reported below eachblot. Values represent means+SEM, N=3. * p<0.05 compared to HPrEC.

Using proteomic analysis, peptides from FLNA, FLNB and KRT19 were alsodetected in cell culture media conditioned by prostate cancer cells (24h), indicating that they can be secreted (FIG. 32). Specifically,conditioned media from LnCAP, DU145, and PC-3 cells exposed to lypoxia(1% oxygen), TNFα (10 ng/mL), or R1881 (1 nM) for 24 hours washarvested, and proteomic analysis was performed. Values representmeans+SEM, N=3. * p<0.05 compared to normoxia control.

Importantly, unlike PSA expression, global regulation of FLNA, FLNB, andKRT19 expression remained unaltered after treatment with multipleprostate-cancer relevant stimula (e.g., hypoxia, androgens, andinflammatory stimula) (FIGS. 33-35). Specifically, mRNA was preparedfrom LnCAP, DU145, and PC-3 cells exposed to hypoxia (1% oxygen; A),TNFα (10 ng/mL; B), or R1881 (1 nM; C) for 24 h. Expression of FLNA,FLNB, and KRT19 was assessed by quantitative RT-PCR, normalized to TBP,and compared to PSA. Values represent means+SEM, N=3. * p<0.05 comparedto normoxia/control.

Assessment of plasma FLNA and FLNB levels as screening markers wereassessed in a proof-of-concept sample cohort of 47 plasma samples (FIG.36). Residual lithium herpain plasma was collected from patient samplesafter the ordered tests were completed. The inclusion criteria wereelevated PSA results (>2.6 ng/mL), age of 45-70 years, and minimumvolume of 700 μL. Results demonstrate that FLNA and FLNB were detectedin human plasma and have predictive power in identifying prostate cancerpatients.

Finally, in vivo validation was next conducted in sera from men (N-447)with confirmed prostate cancer, benign prostate tumors, or BPH using LDTELISA assays in a CLIA-certified laboratory. To assess the sensitivityand specificity of FLNA, FLNB, and KRT19 compared to PSA, ROC curveanalysis was performed. The individual predictive power of eachbiomarker alone was comparable to that of PSA. However, the combinationof age, levels of FLNA, FLNB, and KRT19 and PSA out-performed PSA alonein identification of patients with prostate cancer stratified comparedto benign status, Gleason scores, and incidence of BPH. Together, thesedata indicate that FLNA, FLNB, and KRT19 can be used in conjunction withPSA and/or age for more sensitive and specific prostate cancerscreening, a critical unmet need in the field.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments and methods described herein. Such equivalents are intendedto be encompassed by the scope of the following claims.

It is understood that the detailed examples and embodiments describedherein are given by way of example for illustrative purposes only, andare in no way considered to be limiting to the invention. Variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are included within the spirit and purview ofthis application and are considered within the scope of the appendedclaims. For example, the relative quantities of the ingredients may bevaried to optimize the desired effects, additional ingredients may beadded, and/or similar ingredients may be substituted for one or more ofthe ingredients described. Additional advantageous features andfunctionalities associated with the systems, methods, and processes ofthe present invention will be apparent from the appended claims.Moreover, those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.

1. A method for detecting the level of filamin A in a sample from asubject comprising: (a) selecting a subject who has elevated PSA and issymptomatic of prostate cancer; (b) isolating a serum sample or a plasmasample from the subject, and (c) detecting the protein level of filaminA in the serum sample or plasma sample.
 2. The method of claim 1,further comprising detecting the level of one or more additional markersof prostate cancer in the sample.
 3. The method of claim 2, wherein theone or more additional markers of prostate cancer is selected from thegroup consisting of filamin B, LY9, keratin 4, keratin 7, keratin 8,keratin 15, keratin 18, keratin 19, tubulin-beta 3, and prostatespecific antigen (PSA).
 4. (canceled)
 5. The method of claim 96, whereinthe reagent is an anti-filamin A antibody that selectively binds to atleast one epitope of filamin A.
 6. The method of claim 5, wherein thelevel of filamin A protein is determined by immunoassay or ELISA. 7.(canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled)
 11. (canceled) 12.(canceled)
 13. (canceled)
 14. (canceled)
 15. A method for detecting thelevel of filamin A in a sample from a subject comprising: (a) obtaininga serum sample or a plasma sample from a subject, wherein the subjecthas elevated PSA and is symptomatic of prostate cancer, and (b)detecting the protein level of filamin A in the serum sample or plasmasample.
 16. The method of claim 97, wherein the reagent is ananti-filamin A antibody that selectively binds to at least one epitopeof filamin A.
 17. The method of claim 16, wherein the antibody comprisesa detectable label.
 18. The method of claim 16, wherein the step ofdetecting the level of the complex further comprises contacting thecomplex with a detectable secondary antibody and measuring the level ofthe secondary antibody.
 19. The method of claim 15, further comprisingdetecting the level of one or more additional markers of prostatecancer.
 20. The method of claim 19, wherein the one or more additionalmarkers of prostate cancer is selected from the group consisting offilamin B, LY9, keratin 4, keratin 7, keratin 8, keratin 15, keratin 18,keratin 19, tubulin-beta 3, and prostate specific antigen (PSA). 21.(canceled)
 22. (canceled)
 23. (canceled)
 24. The method of claim 16,wherein the level of the complex is detected by immunoassay or ELISA.25.-67.
 68. A kit for detecting filamin A in a biological sample from asubject having, suspected of having, or at risk for having prostatecancer comprising at least one reagent for measuring the level offilamin A in the biological sample from the subject, and a set ofinstructions for measuring the level of filamin A in the biologicalsample from the subject. 69-86. (canceled)
 87. The method of any one ofclaims 1-3, 5, 6, 15-20, 24, and 94-97, further comprising determiningthe age of the subject. 88.-93. (canceled)
 94. The method of claim 5,wherein the antibody comprises a detectable label.
 95. The method ofclaim 5, wherein the step of detecting the level of the complex furthercomprises contacting the complex with a detectable secondary antibodyand measuring the level of the secondary antibody.
 96. The method ofclaim 1, wherein detecting the protein level of filamin A in the serumsample or plasma sample is by contacting the sample with a reagent thatselectively binds keratin A, allowing a complex to form between thereagent and filamin A, and detecting the level of the complex.
 97. Themethod of claim 15, wherein detecting the protein level of filamin A inthe serum sample or plasma sample is by contacting the sample with areagent that selectively binds keratin A, allowing a complex to formbetween the reagent and filamin A, and detecting the level of thecomplex.